Device and methods of using device for detection of hyperammonemia

ABSTRACT

The present disclosure relates to a biosensor capable of measuring the total concentration of one or a plurality of ammonia or ammonium ions with the use of indophenol reagents in the presence of an ionomer. In some embodiments, the biosensor comprises a perflurinated membrane that comprises an ionomer in contact with an alkali buffer in a vessel configured to receive a sample, such as whole blood. The disclosure also relates to a method of detecting or quantifying the ammonia or ammonium ion concentration in whole blood in a point of care bio sensor without reliance on gas chromatography or any measurement that takes more than about twenty minutes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is an international application designating the UnitedStates of America and filed under 35 U.S.C. § 120, which claims priorityto U.S. Provisional Ser. No. 61/872,149, filed on Aug. 30, 2013, whichis herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This disclosure was made jointly by the NIH and with government supportunder HHSN268201200360P awarded by the NIH. The United States governmenthas certain rights in the disclosure.

FIELD OF THE DISCLOSURE

The disclosure relates generally to devices that quantify and identifythe presence or absence of ammonia or ammonimum ion in a sample ofbodily fluid, water, or other environmental sample. In some embodiments,the disclosure relates to diagnosing a subject with an hyperammonemia bydetecting the presence, absence, or quantity of ammonia or ammonium ionin a sample of bodily fluid. In some embodiments, the device is abiosensor only requiring a sample of whole bodily fluid for detectionand/or quantification of ammonia or ammonium ion.

BACKGROUND OF THE DISCLOSURE

Elevated ammoma levels, oftentimes called hyperammonemia, is apotentially fatal symptom associated with a variety of diseases such ascirrhosis of the liver and urea cycle disorders found in neonatalinfants. Left untreated, hyperammonemia can lead to cognitivedevelopmental issues, seizures, other neurological problems, and death.The current testing methods include fluorometry and tandem massspectroscopy performed by central laboratories, which could takemultiple days to produce a reliable diagnosis. These methods involvelarge, cumbersome, and expensive machinery, which prevents testing ofammonia levels at the bedside or home once the disorder has beenidentified. Therefore, a system for a point of care testing device maybe desired, as this may allow administration of treatment to occur morerapidly, in turn improving the neurological development of infants aswell as making cirrhosis more manageable. Devices able to test forhyperammonemia may also be modified inexpensively to detect amino acidlevels for applications in diagnosing and treating aminoacidopathies andother diseases.

SUMMARY OF DISCLOSURE

The present disclosure encompasses the recognition that hyperammonemiacan be identified and/or characterized by identifying the levels orquantities of ammonia or ammonium ion in any sample, including a bodilyfluid including human and non-human whole blood samples. In someembodiments, the present disclosure relates to identifying the quantity,presence, or absence of ammonia or ammonium ion in bodily fluids bycontacting a bodily fluid to a device disclosed herein. In someembodiments, the methods disclosed herein do not comprise contacting thebodily fluid with any reagent or external stimuli prior to identifyingor quantifying whether or how much one or more ammonia or ammonium ionare present in the bodily fluid.

According to at least one exemplary embodiment, a system, method, andapparatus for point of care hyperammonemia sensors may be disclosed. Thesystem may utilize a phenol, 2-phenylphenol, ninhydrin, potassiumtetraiodomercurate(II), nitroprusside, sodium hydroxide, similarreagents, catalysts, and buffers, or a combination thereof. The systemmay also utilize hyohalite, chloramine T, bleach, or similar chemical.Oftentimes called Berthelot's Reaction or an indophenol reaction, thisreaction may determine ammonia levels in various mediums by changingcolor upon detection ammonia concentration. This may be useful formedical systems, such as in diagnosing hyperammonemia and variousaminoacidopathies; for civil engineering systems, such as in determiningammonia levels of wastewater treatment plants; or for home basedsystems, such as ammonia detection in aquariums or pipes.

According to at least one exemplary embodiment, an apparatus for pointof care hyperammonemia sensors may be disclosed. The apparatus used mayhave a concavity, a fossa, or any other type of well as desired for theplacement of the reagents and sample to be tested. Separating the sampleand reagents may be a cation exchange membrane filter, such as Nafion orsimilar perfluorinated ionomers, to allow the passage of ammonia betweenthe two sections of the well. Anion exchange membranes may also be used,as well as various polymeric hydrogels such as acrylamide, poly(ethyleneglycol) diacrylate, poly(2-hydroxylethyl methacrylate), or poly(vinylalcohol). Additionally, other exemplary embodiments may includemechanisms for quantitative analysis of the color change by means ofphotodiodes and sensors or microfluidic devices that require smalleramounts of reagent and samples.

The present disclosure relates to a biosensor capable of measuring thetotal concentration of ammonia or ammonium ion in a sample with the useof a system comprising reagents for an indophenol or Berthelot reaction,such as hypchlorite, phenylphenol, a basic aqueous solution such asNaOH, and an alkali such as sodium acetate. In some embodiments, thesensor or system comprises at least a first vessel comprising a basicbuffer in aqueous or dried phase. In some embodiments, comprises atleast a first vessel comprises a gel or hydrogel that comprises at leastone or a combination of: an indophenoal reactant or reactants in driedor aqueous phase, a basic buffer in aqueous or dried phase, a alkalisolution in aqueos or dried phase, and or an enzyme that oxidizes atleast one amino acid substrate. The disclosure provides an ammonia orammonium ion biosensor for measuring the total concentration of ammoniaor ammonium ion. In some embodiments, the detection or quantification ofammonia or ammonium ion is accomplished through colorimetric analysiswhereby the reaction products of ammonia or ammonium ion are capable ofemitting a wavelength in the visible spectrum of light. In someembodiments, the system and/or biosensor comprises a diode configured toemit light in at least one vessel and a spectrophotometer configured toreceive light emitted in a vessel containing indophenol or Berthelotreaction reactant products.

In some embodiments, the system and/or biosensor also detects theabsence, presence or quantity of amino acids in solution. In someembodiments, the system and/or biosensor comprises at least a firstelectrically conductive surface (for measuring) and at least a secondelectrically conductive surface (counter electrode), wherein the firstelectrically conductive surface having one or more indophenol reactionreagents described herein or a combination of any one or more indophenolreaction reagents described herein and any one or combination ofconstituent factors, mediators, one or a plurality of enzymes, wherein,if the device comprise one or more enzymes, the one or more enzymesselectively utililize one or more amino acids as substrates. In thoseembodiments with at least a first or second electrode, the one orplurality of enzymes produce reaction products by reacting with thespecified amino acids as substrates, wherein the mediators transportelectrons between the reaction products and the electrode measures aminoacid concentrations, and wherein applied voltages at measuring betweenthe first and second electrically conductive surfaces include such anapplied voltage that, on a working curve representing the relationshipbetween current value and applied voltage with respect to each of theone or plurality of specified amino acids, the distribution of currentvalue at unchanged applied voltage as to individual amino acids. Inother embodiments in which the device, system, and/or bio sensorcomprise at least a first and/or second electrode, the first and/orsecond electrodes are positioned in, substantially adjacent to, oradjacent to at least one vessel in which an indophenol reagent decribedherein may react with one or more components of the reagents. In someembodiments, ammonia of ammonium ion may be the reaction product of oneof the enyxmatic reactions in which the indophenol reaction, using aphenol or phenol related campoud, can take place

According to at least one exemplary embodiment, an apparatus, device,and/or system for point of care hyperammonemia sensors is disclosed. Theapparatus comprises at least a first vessel, or a concavity, a fossa, orany other type of well as desired for the placement of the reagents andsample to be tested. The first vessel may be bifurcated by a membranedisclosed herein or the first vessel may be immediately adjacent to asecond vessel in fluid communication with the second vessel via a fluidexchange opening. In some embodiments, a membrane is positioned at thefluid exchange opening. In some embodiments, the membrane is capable oftransporting ions from the first vessel to the second vessel or viceversa. In some embodiments, the membrane is a cation exchange membranefilter, such as Nafion® or similar membrane comprising perfluorinatedionomers. The membrane allows the passage of ammonia between the twovessels or between the two bifurcated sections of the at least firstvessel. Anion exchange membranes may also be used, as well as variouspolymeric hydrogels such as acrylamide, poly(ethylene glycol)diacrylate, poly(2-hydroxylethylmethacrylate), or poly(vinyl alcohol).

Other exemplary embodiments may include methods and mechnisms forquantitative analysis of ammonia or ammonium ion concentration in asample by contacting a sample to a vessel comprising at least oneindophenol reagent and/or a basic buffer, in either a solid or liquidphase, a section of the vessel exposed to at least a portion of amembrane disclosed herein. In some embodiments, the method comprisesdetecting or quantitating the intensity of a color change within atleast the first or second vessel before and after addition of a sampleto the vessel or vessel. In some embodiments, the method comprisescontacting sample to at least a first vessel, a section or portionexposed or covered by at least one membrane disclosed herein, such firstvessel also optionally comprising at least one indophenol reagentdisclosed herein and/or a basic buffer, either in solid or liquid phase.In some embodiments, if the at least first vessel comprises a buffer,the buffer may be an alkali solution such as sodium acetate or calciumacetate. In some embodiments, the disclosure relates to a method ofcontacting a sample to the device, biosensor or system disclosed hereincomprising at least a first and second vessel, said method comprisescontacting or exposing the sample to the basic buffer in the at leastfirst vessel, allowing ammonia from the sample to transfer to the secondvessel comprising the indophenol reagents disclosed herein. In someembodiments, the disclosure relates to a method of contacting a sampleto the device, biosensor or system disclosed herein comprising at leasta first and second vessel, said method comprises contacting or exposingthe sample to the alkali solution in the at least first vessel, allowingammonia from the sample to transfer to the second vessel comprising theindophenol reagents disclosed herein, the second vessel comprising oneor a plurality of indophenol reactants, which after coming incontactwith the ammonia produce a indephonel or indophenol related compound, Insome embodiments, the contents of the second vessel are exposed to lightmeasue aborbance of light by indphenol compound or indophenol relatedcompound at specific visible wavelengths of light, the absorbance isindicative of or proportionate to a quantity of ammonia or ammonium ionin the sample and whose absorbance is dectected by an individualperforming the test or by a device that measures wavelengths which isincorporated in the device, biosensor, or system disclosed herein. Insome embodiments, the method comprises comparing the absorbance of thecolor or wavelength to a standard which indicates the degree or severityof a hpeyammonemia. In some embodiments, the method comprises contactinga sample to a device, biosensor, or system disclosed comprising a diode,phtodiode, and/or spectrophotometer or other device capable of measuringthe aborbance of wavelength by the indophenol or indophenol relatedcompounds produced as a product of an indophenol reaction within thedevice and exposed to a light. In some embodiments, the deivce,biosensor, and/or system comprise a microfluidic circuit that comprisesat least one conduit configured to receive the sample from a pointexternal to the device, biosensor, and/or system, such microfluidiccircuit comprises a conduit or seris of conduits in fluid communicationwith at least the first and/or second vessel and the one or combinationof: a spectrophotometer, diode, or other device capable of measuring theabsorbance of specific wavelengths by the indophenol or indophenolrelated compound upon its exposure to light.

In some embodiments, the discosure relates to contacting or exposing asample with an alkali buffer and/or a membrane disclosed herein within avessel attached to an electrode able to measure the electron flowproduced by indophenol or an indophenol related compound or redoxtransformation of the metabolite being analyzed. The concentration ofammonia and or ammonium ion and/or metabolite in blood correlates withthe electron flow or current measurments on the circuit that comprisethe at least one electrically conductive surfaces. The disclosurerelates to the reduction to practice of this concept, showing how toselect the metabolite, how to choose an immobilized enzyme, how to dothe immobilization (what polymer, what additives, etc), how to attachthe components to the electrode, how to make a measurement and how dodevelop a prototype. This disclosure is used to measure ammonia orammoniu ion and/or metabolites in blood of patients in real time. Asidefrom the sensor disclosed herein, there are no known sensors able tomeasure the proposed metabolites in real time.

The disclosure also relates to a device or system comprising at leastone electrically conductive surface (such as an electrode) operablyconnected to a diode, a spectrophotometer, voltmeter and/or amperometer,the electrode comprises components that, when combined and in thepresence of an ammonia, cause a indophenol reaction. The indophenolreaction product comprises a molecule that emits a visible or knownwavelength after exposure to light. In some embodiments, the device andsystem disclosed herein comprise a diode, such as a photodiode, whichemits light into the vessel comprising the indophenol reaction productthereby exciting the reaction product and causing the reaction productto emit a visible or known wavelength. In some embodiments, the deviceand system disclosed herein comprise a spectrophotometer that detectsand/or quantitates the intensity of the visible or known wavelength oflight emitted by the indophenol reaction product.

The disclosure also relates to a device and/or system that detects andquantifies amino acids. In some embodiments, the device and/or systemscomprise a vessel or well that comprises a metabolic enzyme disclosedherein or a functional fragment thereof. In some embodiments, theenzymae or fragment thereof is immobilized to the vessel into whichsample is initially place in the device, bio sensor, system, test strip,or catridge. After contact with a sample, the enzyme or functionalfragment thereof releases at least one or a series of electrons andammonia, such that ammonia is free in solution and capable of movingbetween a first vessel to a second vessel through a membrane disclosedherein. In some embodiments, the device comprises at least a first andsecond electrically conductive surface, wherein the first electricallyconductive surface comprises a hydrogel comprising an ezyme disclosedherein and the second electrically conductive surface does not comprisea hydrogel or an enzyme; wherein a voltmeter and/or amperometer areconfigured in a circuit such that the voltmeter can detect a voltagedifferential between the first and second electrodes in the presence ofan amino acid and/or wherein the amperometer can detect an increasedcurrent in the first electrode as compared to the second electrode inthe presence of an amino acid. The at least one or a series of electronsare released after one or more enzymes within the hydrogel catalyzes theoxidation of the amino acid in a bodily sample in the presence of theone or more amino acids.

Hydrogel formulations are used to entrap one or more enzymes (thatutilizes the metabolite/analyte as a specific substrate for itsreaction) along with, in some embodiments, a requisite cofactor in closeproximity to the at least first electrode surface, with the hydrogelproviding a simultaneous exclusion of interfering ions andmacromolecules (contained within the patient's blood sample) from theelectrode sensor. The coated electrode is contained within aelectrochemical detection device capable of converting redox equivalentsgenerated by the enzyme reaction into electron flow which in turn ismeasured as a current or voltage differential. Analyte concentration isderived using a calibration curve that correlates amperage or voltagedifferential to concentration of amino acid in the sample of bodilyfluid. In one embodiment, a small volume of whole blood is applied to orammonia from the ahole blood diffuses to a vessel exposed to theelectrode and the result is reported within minutes of the applicationor contact to the electrode. Depending on the exact analyte, specificenzyme(s) and cofactor(s) are incorporated into the electrode in orderto achieve analyte-specific reaction and response. For example, todetect elevated phenylalanine, the enzyme phenylalanine dehydrogenase isimmobilized to the at least one electrically conductive surfaceoptionally contained within a hydrogel.

The disclosure provides a method of sorting a mixture of samples ofbodily fluid comprising: contacting a plurality of bodily fluid samplesto a device or system disclosed herein. In some embodiments, the methodof sorting or cataloguing a mixture of samples of bodily fluid furthercomprises the step of determining one or more concentrations of ammonia,ammonium ion, and/or amino acid in the bodily fluid sample, if inrespect to the ammonia or ammonium ion concentration, based upon thepresence or quantity of indophenol reaction products in one or morevessels or a current value or voltage differential value measured by thedevice; and, if in respect to determining one or more concentrations ofamino acid in solution, based upon a current value or voltagedifferential value measured by the device. In some embodiments, themethod further comprises the step of comparing the one or moreconcentration of ammonia, ammonium ion, and/or amino acid in a sample ofbodily fluid with one or more concentrations of ammonia, ammonium ion,and/or amino acid in sample of bodily fluid obtained from subject whodoes not have or is not suspected of having one or moreaminoacidopathies or hyperammonemia, and cataloging, compiling, oridentifying whether a sample of bodily fluid from a subject has anaminoacidopathy and/or hyperammonemia based upon their similarities ordifferences in concentration value to a sample of bodily fluid from asubject without an aminoacidopathy and/or hyperammonemia. The disclosureprovides a method of diagnosing a subject with an hyperammonemiacomprising: contacting at least one sample of bodily fluid from thesubject to a device or system disclosed herein. In some embodiments, themethod of diagnosing further comprises the step of determining one ormore concentrations of ammonia and/or ammonium ion in a bodily fluidsample based upon a current value, voltage differential value, or apresence or absence of a wavelength of light emitted by an indophenolreaction product, indophenol or an indophenol related compound. In eachcase, the device and or system disclosed herein detects and/or measuresscuh values. In some embodiments, the method further comprises the stepof comparing the one or more concentration of ammonia, ammonium ion,and/or amino acid in the one or more samples from the subject with oneor more concentrations of amino acids in sample of bodily fluid obtainedfrom subject who does not have or is not suspected of having one or moreaminoacidopathies and/or hyperammonemia, identifying whether a sample ofbodily fluid from a subject has an aminoacidopathy and or hyperammonemiabased upon their similarities or differences in concentration value tothe sample of bodily fluid from a subject without an aminoacidopathyand/or hyperammonemia.

The disclosure also provides a method of monitoring the concentrationsof ammonia or ammonium ion in subject over time in a sample of bodilyfluid from a subject diagnosed or suspected as having hyperammonemia,the method comprising: contacting one or more samples of bodily fluidfrom a subject to a device or system disclosed herein and measuring theconcentration of ammonia or ammonium ion of bodily fluid from thesubject at one time point and performing a repeating of the measurementat least once at a different time point. In some embodiments, the methodof monitoring the concentrations of ammonia or ammonium ion in subjectover time in a sample of bodily fluid from a subject diagnosed orsuspected as having hyperammonemia further comprises the step ofcataloguing the concentration values of the ammonia or ammonium ion overtime. In some embodiments, the method further comprises the step ofcomparing the one or more concentration of amino acids from theplurality of samples of bodily fluid from the subject over time and,optionally notifying a subject if the concentration of one or moreammonia or ammonium ion reaches or exceeds or drops below a thresholdvalue that requires medical treatment or modification of diet.

In some embodiments, samples of bodily fluid are isolated from a subjecthaving been diagnosed with or suspected as having hyperammonemia. Forexample, in some embodiments, a sample of bodily fluid such as a urinesample or a blood sample is isolated from the subject. The sample ofbodily fluid is contacted to at least one electrode comprising at leastone enzyme disclosed herein and the amino acid concentration in thesample of bodily fluid is measured based upon the magnitude of thevoltage differential or current detected by the device comprising the atleast one electrode. In further embodiments, method of the presentdisclosure comprises contacting a sample of bodily fluid to at least oneelectrode comprising an immobilized enzyme disclosed herein, measuringthe current or voltage difference between the at least one electrode andan electrically conductive surface that does not comprise an immobilizedenzyme disclosed herein, determining the concentration of one or moreamino acids in the sample of bodily fluid, and optionally, providing areadout of one or more concentration values to a subject from which thesample of bodily fluid was obtained. In further embodiments, method ofdetecting ammonia or ammonium ion comprises contacting a sample ofbodily fluid to at least one vessel comprising an hyohalite, an aqueousbasic solution, and at least one compound comprising a phenyl groupdisclosed herein, measuring the current or voltage difference betweenthe at least one electrode and an electrically conductive surface thatdoes not comprise an immobilized enzyme disclosed herein, determiningthe concentration of ammonia or ammonium ion in the sample of bodilyfluid, and optionally, providing a readout of one or more concentrationvalues to a subject from which the sample of bodily fluid was obtained.

In some embodiments, the present disclosure provides methods comprisingcontacting a sample of bodily fluid from a subject to an aqueous basicsolution or a basic buffer in a dried or powdered phase, exposing thesample to hyohalite and at least one compound comprising a phenyl groupin the presence (or absence—to establish a control value) of a membranecomprising an ionomer, and optionally contacting a gel. In someembodiments, the gel is a hydrogel comprising alginate. In someembodiments, the present disclosure provides methods comprisingdetecting presence or level ammonia or ammonium ion in a sample ofbodily fluid between cells in the sample. In some embodiments, providedmethods comprise determining that a particular set of detectedinteractions defines an threshold value (or control value) that ischaracteristic of an increased severity of hyperammonemia in that itdistinguishes them from elevated or non-elevated amino acid levels inanother sample of bodily fluid from the subject or from a sample ofbodily fluid that is a reference or control sample such that, if thethreshold value is reached, the device or system disclosed hereinprovides the subject with a signal or notification that treatment ordiet modification should be sought. In some embodiments, the step ofdetecting comprises detecting presence or level of ammonia or ammoniumion concentrations in a sample of bodily fluid that is characteristic ofparticular severity of disease in the sample in that it distinguishesthem from a sample of bodily fluid that is a reference or controlsample.

In some embodiments, the step of detecting comprises detecting presenceor level of ammonia or ammonium ion concentrations in a sample of bodilyfluid that is characteristic of particular severity of disease in thesample in that it distinguishes them from a sample of bodily fluid thatis a reference or control sample.

In some embodiments, any of the methods disclosed herein do not comprisepre-treating the sample of bodily fluid prior to contacting the samplewith the test strip, conduit, biosensor, and/or at least oneelectrically conductive surface. In some embodiments, any of the methodsdisclosed herein do not comprise using at step of treating the samplewith liquid chromatography, gas chromatography, and/or electrophoresisbefore, simultaneously with or after contacting the sample to the teststrip, conduit, biosensor, and/or at least one electrically conductivesurface. In some embodiments, any of the methods disclosed hereincomprise contacting the sample to at least one electrode that does notcomprise an enzyme obtained from an organism other than a bacteria or aplant.

The disclosure relates to methods of detecting the levels of ammonia orammonium ion in whole blood by exposing a whole blood sample to one ofthe biosensors, systems, or devices disclosed herein. The disclosurealso relates to manufacturing a biosensor disclosed herein by treatingthe membrane with one, two, three or more washes of an acidic solutionprior to placement of the membrane at a fluid exchange opening or at avessel. The disclosure relates to manufacturing a biosensor disclosedherein by treating the membrane with one, two, three or more washes ofan acidic solution from about 0.1 M to about 1 M H₂SO₄ prior toplacement of the membrane at a fluid exchange opening or at a vessel.The disclosure also relates to manufacturing a biosensor disclosedherein by treating the membrane with one, two, three or more washes ofan hydrogen peroxide solution from about 0.1 M to about 1 M H₂O₂ priorto placement of the membrane at a fluid exchange opening or at a vessel.The disclosure relates to manufacturing a biosensor disclosed herein bytreating the membrane with one, two, three or more washes of an acidsolution and/or a hydrogen proxide solution prior to placement of themembrane at a fluid exchange opening or at a vessel. The disclosure alsorelates to manufacturing a biosensor disclosed herein by treating themembrane with one, two, three or more washes of an acid solution fromabout 0.1 M to about 1 M H₂O₂ prior to placement of the membrane at afluid exchange opening or at a vessel. The disclosure also relates tomanufacturing a biosensor disclosed herein by treating the membrane withone, two, three or more of an acid solution comprising from about 0.1 Mto about 1 M H₂SO₄ and with one, two, three or more washes of anhydrogen peroxide solution comprising from about 0.1 M to about 1 M H₂O₂prior to placement of the membrane at a fluid exchange opening or at avessel.

In some embodiments, the present disclosure provides a system comprisingone or more devices disclosed herein optionally in operable connectionto a electronic storage medium that compiles ammonia or ammonium ionand/or amino acid concentration values of a subject. In someembodiments, the electronic storage medium comprises compiled amino acidconcentration values of a subject over time. In some embodiments, thesystem comprises at least one electrically conductive surface thatcomprises an enzyme disclosed herein, a mediator, and optionally a gelor hydrogel. In some embodiments, the system comprises an electroniccircuit that is in operable connection to the at least one electrodesand a diode, spectrophotometer, voltmeter and/or amperometer. In thecase of a diode and/or spectrophotometer, the diode or spectrophotometerdetect and wavelength of light emitted from the at least one vessel. Inthe case of the voltmeter and/or amperometer, the voltmeter and/oramperometer measures the respective voltage and/or amperage of thecircuit across the at least one electrode when the at least oneelectrode is in the presence of one or more amino acids and/or ammoniaconcentrations. In some embodiments, system comprising one or moredevices disclosed herein optionally in operable connection to aelectronic storage medium that compiles amino acid concentration valuesof a subject determines one or a plurality of concentration values ofammonia concentration values and/or amino acids in a sample of bodilyfluid when the sample of bodily fluid is in contact with the at leastone electrode and under conditions and for a time sufficient for for theindophenol reaction to take place or the one or more enzymes disclosedherein to oxidize its amino acid substrate, create a voltagedifferential or current change in the circuit and the device to displaythe concentration value on one or more displays. In some embodiments,the device, system, and/or biosensor do not comprises one or moreelectrodes.

In some embodiments, the disclosure provides for a method comprisingsteps of: contacting a sample comprising cells with an electrode. Thedisclosure further provides for a method comprising steps of: contactinga sample under conditions and for a time sufficient for a set ofinteractions to occur between ammonia in a sample and the membranedescribed herein. The disclosure further provides for a methodcomprising steps of: contacting a whole blood sample under conditionsand for a time sufficient for a set of interactions to occur betweenammonia in a sample and the membrane described herein. The disclosurefurther provides for a method comprising steps of: contacting a samplecomprising bodily fluid under conditions and for a time sufficient for aset of interactions to occur between the ammonia in the sample and theone or plurality of indophenol reaction reagents described herein.

The disclosure relates to a biosensor comprising: at least oneelectrically conductive support, the electrically conductive supportattached to a hydrogel, the hydrogel comprising at least one electronmediator, at least one reduction agent, and at least one metabolicenzyme or functional fragment thereof, wherein the hydrogel comprisesalginate; and an amperometer and/or voltmeter operably connected to theat least one electrically conductive support or surface.

In some embodiments, the biosensor comprises at least three electricallyconductive supports. In some embodiments, the at least one electricallyconductive support is a silver and silver chloride wire. In someembodiments, the at least one electrically conductive support comprisesat least one or a combination of metabolic enzymes chosen from: leucinedehydrogenase, tyrosine dehydrogenase, phenylalanine dehydrogenase,leucine oxidoreductase, tyrosine monooxygenase, alanine dehydrogenase,or glutamate dehydrogenase; or functional fragments thereof. In someembodiments, the biosensor comprises at least a first and a secondelectrically conductive support, wherein the first electricallyconductive support is attached to a hydrogel, the hydrogel comprising atleast one electron mediator, at least one reduction agent, and at leastone metabolic enzyme or functional fragment thereof, wherein said firstand second electrically conductive supports being operably connected tosaid voltmeter and/or amperometer to apply a voltage therebetween.

In some embodiments, the at least one electrically conductive supportcomprises an electronegative or anionic chemical component. In someembodiments, the at least one hydrogel comprises trehalose. In someembodiments, the biosensor does not comprise one or more of thefollowing: (i) uricase or a functional fragment thereof; (ii) a hydrogelcomprising dextran or a derivative thereof; (iii) a bacterial cell; (iv)an electronic dipole configured for electrophoresis; and (v) 3, 4-DHB.In some embodiments, the biosensor is at least 70% biologically activeafter about sixteen days in storage at 4 degrees Celsius. In someembodiments, the biosensor is at least 70% biologically active afterabout thirty days in storage at 4 degrees Celsius. In some embodiments,the biosensor is at least 80% biologically active after about thirtydays in storage at 4 degrees Celsius In some embodiments, the biosensoris at least 90% biologically active after about thirty days in storageat 4 degrees Celsius In some embodiments, the biosensor is at least 95%biologically active after about thirty days in storage at 4 degreesCelsius. In some embodiments, the biosensor is not functionallydependent upon exposure to UV light or addition of any stimulus externalto the biosensor. In some embodiments, the at least one enzyme orfunctional fragment thereof is derived from a bacterial species and isimmobilized in the hydrogel. In some embodiments, the at least oneenzyme or functional fragment thereof is derived from a thermophillicbacterial species and is immobilized in the hydrogel. In someembodiments, the at least one enzyme or functional fragment thereofcomprises at least about 70% sequence identity to SEQ ID NO:1 or SEQ IDNO:2.

In some embodiments, the disclosure relates to a biosensor, device, orsystem disclosed herein comprise a circuit comprising at least a firstand second electrode in electric communication to at least one or acombination of a diode, photodiode, spectrophotometer, or other devicecapable of measuring the presence, absence, or intensity of lightemitted by an amount of indophenol or indophenol relate compound exposedto light. In some embodidments the cioruit comprises a wire. In someembodiments, the wire comprises silver and silver chloride in operableconnection to the voltmeter and/or amperometer.

In some embodiments, the biosensor, device, and/or system disclosedherein comprises a membrane optionally comprsing alginate comprises ablock polymer with a formula

wherein m and n are any positive integer.

In some embodiments, the biosensor the at least one electricallyconductive support is not covered by a membrane comprising cellulose ora derivative thereof. In some embodiments, the at least one electronmediator is selected from: thionine, o-phenylenediamine, methylene blue,and toluidine blue. In some embodiments, the at least one reductionagent is chosen from: NAD+ or FAD+.

The disclosure also relates to a biosensor comprising: at least oneelectrically conductive support, the electrically conductive supportattached to at least one hydrogel, the hydrogel comprising at least oneelectron mediator, at least one reduction agent, and at least onemetabolic enzyme or functional fragment thereof; wherein the at leastone enzyme or functional fragment thereof is at least 70% homologous toa phenylalanine dehydrogenase from Geobacillus thermoglucosidiasus; andan amperometer and/or voltmeter operably connected to the at least oneelectrically conductive support. In some embodiments, the enzyme orfunctional fragment thereof is at least 70%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% homologous to SEQ ID NO:1 or at least 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% homologous to a functional fragment of SEQ IDNO:1. In some embodiments, the enzyme or functional fragment thereof isnot derived from a species other than a bacterial cell. In someembodiments, the enzyme or functional fragment thereof is at least 70%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homologous to SEQ ID NO:2 or atleast 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homologous to afunctional fragment of SEQ ID NO:2.

The disclosure relates to a system comprising a bio sensor comprising atleast a first and second vessel; a fluid exchange opening positionedbetween the at least first and second vessel; at least one conduit influid communication with the at least first vessel, the at least oneconduit configured to receive a fluid from a point external to thebiosensor; and a membrane positioned at the fluid exchange opening;wherein the membrane comprises an ionomer, and wherein the first vesselor the second vessel comprise, individually or in combination:hyohalite, an aqueous basic solution, and at least one compoundcomprising a phenyl group.

The disclosure also relates to a system comprising a biosensor disclosedherein optionally comprising an electric circuit comprising any one orcombination of: a diode (such as a photodiode), a spectrophotometer, anamperometer and/or voltmeter operably connected to the at least oneelectrically conductive support or surface; wherein the bio sensor is inoperable connection to at least one computer storage memory. In someembodiments, the system further comprises a sample of bodily fluid, suchas whole blood. In some embodiments, the system further comprises adigital display in operable connection to the at least one electricallyconductive support (or surface) by an electrical circuit capable ofcarrying an a electrical signal corresponding to a measurement ofcurrent and/or voltage differential from the diode, a spectrophotometer,voltmeter and/or amperometer to the digital display, wherein the digitaldisplay is a configured to display one or more concentration values ofammonia or ammonium ion and/or an amino acid in a sample over time whenthe at least one electrically conductive support (or surface) is incontact with the sample for a time period sufficient for the indophenolreaction to take place.

In some embodiments, the system further comprises a computer processorin operable connection with the at least one computer storage memory. Insome embodiments, the metabolic enzyme is a phenylalanine dehydrogenaseimmobilized within the hydrogel and wherein the alginate concentrationof the hydrogel is from about 1% to about 3% weight to volume of thetotal volume attached and/or contatcted to the at least one electricallyconductive support.

The disclosure also relates to a kit comprising a biosensor comprising adiode, spectrophotometer, voltmeter and/or amperometer and a displayconfigured in an electrical circuit such that, upon contact with atleast one removable electrically conductive support, the circuit becomesclosed such that the diode, spectrophotometer, voltmeter and/oramperometer are in operable communication with at least one electricallyconductive support.

In some embodiments, the kit comprises at least one of the following: aplurality of test strips comprising one or a plurality of vesselsconfigured to receive a sample, such as whole blood, wherein the one orplurality of test strips further comprises at least one conduit in fluidcommunication with the at least first vessel. In some embodiments, thethe kit comprises at least one of the following: a plurality of teststrips comprising one or a plurality of vessels configured to receive asample, such as whole blood, wherein the one or plurality of test stripsfurther comprises at least one conduit in fluid communication with theat least first vessel and, individually or in combination: hyohalite, anaqueous basic solution, and at least one compound comprising a phenylgroup. In some embodiments, the kit comprises at least one of thefollowing: a plurality of test strips comprising one or a plurality ofvessels configured to receive a sample, such as whole blood, wherein theone or plurality of test strips further comprises at least one conduitin fluid communication with the at least first vessel and a biosensorcomprising a membrane disclosed herein. In some embodiments, the kitcomprises at least one of the following: a plurality of test stripscomprising: one or a plurality of vessels configured to receive asample, such as whole blood, wherein the one or plurality of test stripsfurther comprises at least one conduit in fluid communication with theat least first vessel; and a biosensor comprising a membrane disclosedherein.

In some embodiments, the membrane comprises a hydrogel layer. In someembodiments, the hydrogel layer comprises alginate. In some embodiments,the a control or reference sample of bodily fluid; a set of datacomprising threshold values; and a set of instructions, wherein the setof instructions or the set of data optionally accessible remotelythrough an electronic medium. In some embodiments, the kit comprises asolid support that comprises at least a first and a second electrode,wherein the first electrode comprises a hydrogel, the hydrogelcomprising at least one electron mediator, at least one reduction agent,and at least one metabolic enzyme or functional fragment thereof; andwherein the second electrode is a control or reference electrode. Insome embodiments, the kit comprises a test strip comprising a solidsupport attached to a first and a second electrode described herein.

The disclosure also relates to a method of determining or identifying aconcentration of an ammonia or ammonium ion in a sample of bodily fluidcomprising: (a) contacting a sample of bodily fluid to: (i) a biosensorcomprising at least one electrically conductive support, theelectrically conductive support attached to a vessel in fluidcommunication with a membrane disclosed herein and, optionally anamperometer and/or voltmeter operably connected to the at least oneelectrically conductive support; or (ii) a system comprising a biosensorcomprising: at least one electrically conductive support attached to avessel in fluid communication with a membrane disclosed herein and,optionally an amperometer and/or voltmeter operably connected to the atleast one electrically conductive support; or (iii) a test stripdisclosed herein; and/or (b) determining a quantity of ammonia orammonimum ion in the sample. In some embodiments, the sample of bodilyfluid comprises blood or serum from a subject. In some embodiments, thesample consists of whole blood or consists essentially of whole blood.

The disclosure also relates to a method of quantifying a concentrationof ammonia and/or an amino acid in a sample of bodily fluid comprising:(a) contacting a sample of bodily fluid to: (i) a biosensor comprisingat least one electrically conductive support or surface, theelectrically conductive support or surface attached to a hydrogel, thehydrogel comprising at least one electron mediator, at least onereduction agent, and at least one metabolic enzyme or functionalfragment thereof, wherein the hydrogel comprises alginate; and anamperometer and/or voltmeter operably connected to the at least oneelectrically conductive support or surface; or (ii) a system comprisinga biosensor comprising: at least one electrically conductive support orsurface, the electrically conductive support or surface attached to atleast one hydrogel, the hydrogel comprising at least one electronmediator, at least one reduction agent, and at least one metabolicenzyme or functional fragment thereof; wherein the at least one enzymeor functional fragment thereof is at least 70% homologous to aphenylalanine dehydrogenase from Geobacillus thermoglucosidiasus; and anamperometer and/or voltmeter operably connected to the at least oneelectrically conductive support; or (iii) a test strip disclosed herein;or (b) determining a quantity of amino acid in the sample. In someembodiments, the method further comprises comparing a concentrationvalue obtained by the quantifying or identifying steps to a thresholdvalue associated with one or more metabolic diseases.

The disclosure further relates to a method comprising a step ofcontacting a biosensor, system, or test strip disclosed herein, whereinthe step of contacting a sample of bodily fluid of a subject to any ofthe disclosed biosensors, systems, or test strips comprises contactingthe sample for a sufficient period of time to allow ammonia transportthrough the membrane and to expose the ammonia from the sample toreagents associated with an indophenol reaction. If amino acids are alsobeing tested by the biosensors, systems, or test strips, such methodscomprise contacting a sample for a sufficient period of time to allowoxidation of at least one amino acid in the sample of bodily fluid bythe metabolic enzyme or functional fragment therof. In some embodiments,the method does not comprise exposing the sample of bodily fluid to anyexternal stimuli or reagent prior to contacting the sample to the atleast one electrically conductive supports. In some embodiments, themethod does not comprise exposing the sample of bodily fluid to ironions and/or hydrozide ions prior to, simultaneously with, or afterexposing the sample to the at least one electrode comprising a hydrogel.In some embodiments, the method does not comprise exposing the sample toa non-porous carrier, such as glass beads, contained within the device,test strip or biosensor. In some embodiments, the sample of bodily fluidcontains whole blood or serum from a subject. In some embodiments, thesample of bodily fluid does not contain urine. In some embodiments, thesample of bodily fluid does not contain bodily fluid other than wholeblood or blood serum.

The disclosure further relates to a method of diagnosing a metabolicdisease in a subject comprising: (a) contacting a sample of bodily fluidto one or a combination of: (i) a biosensor comprising at least oneelectrically conductive support or surface, the electrically conductivesupport or surface attached to a vessel comprising an amount ofindophenol or indophenol related compound; and, optionally anamperometer and/or voltmeter operably connected to the at least oneelectrically conductive support or surface; or (ii) a system comprisinga bio sensor comprising: at least one electrically conductive support orsurface, the electrically conductive support or surface exposed to th atleast first vessel or second vessel comprising the indophenol and/orindophenol related compounds; and an amperometer and/or voltmeteroperably connected to the at least one electrically conductive support;or (iii) a test strip disclosed herein; (b) quantifying one or moreconcentration values of ammonia or ammonium ion in the sample; (c)comparing the one or more concentration values of ammonia or ammoniumion in the sample to a threshold value of ammonia or ammonium ionconcentration identified as being in a healthy range or not within arange or concentration indicative of hyerammonemia; and (d) identifyingthe subject as having hyperammonemia or a metabolic disease related tohyperammonemia if the one or more concentration values of amino acids inthe sample exceed or fall below the threshold value. In someembodiments, the metabolic disease is a hyperammonemia related disorder.

The disclosure also relates to a method of determining patientresponsiveness to a therapy comprising: (a) contacting a sample ofbodily fluid to one or a combination of: (i) a bio sensor comprising atleast one electrically conductive support or surface, the electricallyconductive support or surface attached to a vessel comprising an amountof indophenol or indophenol related compound; and, optionally anamperometer and/or voltmeter operably connected to the at least oneelectrically conductive support or surface; or (ii) a system comprisinga bio sensor comprising: at least one electrically conductive support orsurface, the electrically conductive support or surface exposed to th atleast first vessel or second vessel comprising the indophenol and/orindophenol related compounds; and an amperometer and/or voltmeteroperably connected to the at least one electrically conductive support;or (iii) a test strip disclosed herein; (b) quantifying one or moreconcentration values of ammonia or ammonium ion in the sample; (c)comparing the one or more concentration values of ammonia or ammoniumion in the sample to a threshold value of ammonia or ammonium ionconcentration identified as being in a healthy range or not within arange or concentration indicative of hyerammonemia; and (d) identifyingthe subject as having hyperammonemia or a metabolic disease related tohyperammonemia if the one or more concentration values of amino acids inthe sample exceed or fall below the threshold value.

The disclosure also relates to a test strip comprising a solid supportand at least a first and a second electrode attached to the solidsupport, wherein the first electrode comprises a membrane, the membranecomprising a perfluirnated ionomer. In some embodiments, the test stripis adapted for a portable device comprising: a diode, spectrophotometer,voltmeter and/or amperometer; and a digital display such that, when thetest strip is contacted to the device, the first and second electrodesbecome operably connected to a closed electrical circuit comprisingdiode, spectrophotometer, voltmeter and/or amperometer and the digitaldisplay, and, upon contact with light emitted from an indophenol orindophenol related compound, resulting in a current on the firstelectrode corresponding to a concentration value of amino acid in thesample of bodily fluid, such concentration value readable on the displayof the portable device. In some embodiments, the test strip comprisesthe at least one or combination of indophenol reagents in solid orliquid phase optionally separated from but in fluid communication with aconduit, volume, or space the at least first vessel.

The disclosure also relates to a method of manufacturing any of thedisclosed biosensors, test strips, systems disclosed herein thatcomprises at least one electrode, the method comprising: contacting theat least one electrode with a solution comprising at least one vessel,at least one conduit in fluid communication with the at least onevessel, and at least one indophenol reagent; subsequently contacting theat least one electrode with a basic buffer with a Na+, Ca+, Cl−, and/oracetate concentration at or below about 1 M.

BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of embodiments of the present invention will be apparent fromthe following detailed description of the exemplary embodiments. Thefollowing detailed description should be considered in conjunction withthe accompanying figures in which:

FIG. 1 is an exemplary view of a system having the ability to detectammonia or ammonium ion levels in a given sample applied to a first andsecond vessel separated by a membrane positioned at an fluid exchangeopening.

FIG. 2 is an exemplary view of a system comprising multiple vesselswithin which more than one indophenol reaction mat be performed inparallel.

FIG. 3 shows exemplary reaction otherwise known as Berthelot's Reactionor an indophenol reaction.

FIG. 4 shows an exemplary embodiment of a microfluidic testing device.

FIG. 5 shows an exemplary flowchart for a method of quantitative pointof care hyperammonemia sensing using embodiments of the disclosure.

FIG. 6 shows an exemplary embodiment of a blood test strip for use withan electronic testing device.

FIG. 7 shows an exemplary embodiment of a device comprising anelectronic circuit comprising an electrode exposed to a vesselconfigured for performance of the indophenol reaction; an analog todigital convertor, a microchip in electronic communication with adisplay.

FIG. 8 shows the chemical composition of Nafion.

FIG. 9 shows experimental data demonstrating how the concentration ofsodium salt yields high recovery and transfer of ammonia from a sample.

FIG. 10 shows experimental data demonstrating the differences in deviceperformance when using sodium acetate versus sodium chloride as a basicbuffer.

FIG. 11 depicts a photograph of the 3D printed modular pieces snappedtogether around Nafion to form the bisected well utilized for thesensing experiments

FIG. 12 depicts an indophenol reaction produces a linear curve withconcentrations of ammonium chloride ranging from 0-750 μM with a COD of0.9939.

FIG. 13 depicts reagents for the indophenol reaction were stored at roomtemperature and used to generate an ammonia standard curve at regularintervals for 100 days. The response to 500 μM ammonia began to degradeat day 75. The reagents of the indophenol reaction are stable at roomtemperature for up to 50 days before its response to differentconcentrations of ammonia begins to deteriorate.

FIG. 14 depicts 1 mM concentrations of each of the 21 amino were testedusing the indophenol reaction. The absorbance measured at 635 nm foreach amino acid after the indophenol reaction was calculated aspercentage of the response from indophenol reaction with 1 mM ammoniumchlroide. The radar graph displays the percent response as compared toammonium chloride. The highest response was threonine which produced anabsorbance value that was just 7% of ammonia's response.

FIG. 15: The constructed sensor's response to a range of ammoniaconcentrations in 1×PBS. The COD is 0.9758 with n=5 samples.

FIG. 16: Initial experiments of determining blood ammonia concentrationdemonstrated a limited response. Responses were hindered and would notexceed an absorbance of 0.35 indicating some degree of interference.

FIG. 17: Concentrations of 2-10× hypochlorite were utilized in theanalysis of 500 mM ammonia in 1×PBS and whole sheep's blood. Increasingthe concentration of hypochlorite utilized in the indophenol reactionreduced the negative interference small blood molecules had on theindophenol reaction. At concentrations higher than 3×, reaction itselfbegan to degrade. A 3-fold increase in hypochlorite concentration wasoptimal.

FIG. 18: The bisected well sensor was again used to extract ammonia inwhole human blood. The extracted ammonia solutions were tested with the3× hypochlorite-modifed indophenol reaction and the absorbance measuredat 635 nm. In the range of 0-500 μM the COD was 0.9573 with n=5 samples.

FIG. 19: The sensor's response to blood ammonia concentrations rangingfrom 0-150 μM. The relative standard deviation is ˜10% with a COD of0.9777 with n=5 samples.

FIG. 20: depicts a CAD sketch of the front face of the well plate of anembodiment.

FIG. 21: depicts a CAD sketch demonstrating the area of the well plateof an embodiment that the adhesive silicone should be attached to (blackarea) prior to completing the manufacturing of the device.

FIG. 22 depicts a photograph of the 3D-printed modular left and rightsides pieces snapped together around Nafion® to divide the well into twosections.

FIG. 23 depicts an engineering drawing of 3D printed well.

FIG. 24 depicts a CAD sketch of the top piece of a disposable catridge,with dimensions in mm. Channels 1 through 5 are labeled.

FIG. 25: CAD sketch of the bottom piece of the chip with channel 6labeled.

FIG. 26: depicts the representations of a concentration profile at i)t=0 seconds (s); ii) t=13 s, and iii) t=24 s after a whole blood samplein 40 microliters is loaded into well number 6.

FIG. 27 depicts the top half of an embodiment comprising a microfluidicdevice used to quantify ammonia levels in whole blood.

FIG. 28 depicts the bottom half of an embodiment comprising amicrofluidic device used to quantify ammonia levels in whole blood.

FIG. 29: is an exemplary view of a system having the ability to detectammonia or ammonium ion levels and amino acids in a given sample appliedto a first and second vessel separated by a membrane positioned at anfluid exchange opening, wherein the reaction is catalyzed by an enzyme.

DETAILED DESCRIPTION OF THE DISCLOSURE

Various terms relating to the methods and other aspects of the presentdisclosure are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

As used herein, the terms “attach,” “attachment,” “adhere,” “adhered,”“adherent,” or like terms generally refer to immobilizing or fixing, forexample, a group, a compound or enzyme, to a surface, such as byphysical absorption, chemical bonding, and like processes, orcombinations thereof.

As used herein, the terms “biopsy” means a cell sample, collection ofcells, or bodily fluid removed from a subject or patient for analysis.In some embodiments, the biopsy is a bone marrow biopsy, punch biopsy,endoscopic biopsy, needle biopsy, shave biopsy, incisional biopsy,excisional biopsy, or surgical resection.

As used herein, the terms “bodily fluid” means any fluid from a isolatedfrom a subject including, but not necessarily limited to, blood sample,serum sample, a whole blood sample, urine sample, mucus sample, salivasample, and sweat sample. The sample may be obtained from a subject byany means such as intravenous puncture, biopsy, swab, capillary draw,lancet, needle aspiration, collection by simple capture of excretedfluid.

As used herein the terms “electronic medium” mean any physical storageemploying electronic technology for access, including a hard disk, ROM,EEPROM, RAM, flash memory, nonvolatile memory, or any substantially andfunctionally equivalent medium. In some embodiments, the softwarestorage may be co-located with the processor implementing an embodimentof the disclosure, or at least a portion of the software storage may beremotely located but accessible when needed.

As used herein, the word “exemplary” means “serving as an example,instance or illustration.” The embodiments described herein are notlimiting, but rather are exemplary only. It should be understood thatthe described embodiment are not necessarily to be construed aspreferred or advantageous over other embodiments. Moreover, the terms“embodiments of the invention”, “embodiments” or “invention” do notrequire that all embodiments of the invention include the discussedfeature, advantage or mode of operation. In addition, those skilled inthe art may appreciate the wide variations in sizing scales that may beincorporated into the disclosed or related designs for use with samplesmany orders of magnitude larger or smaller than those disclosed.

As used herein, the term “aminoacidopathy” is meant to refer to thosediseases and disorders characterized by dysfunction of a metaboliccatalysis of amino acids thate results in over production or underproduction of amino acids in the body of a subject. Examples ofaminoaciopathies are listed in the definition of a metabolic disease,terms that are used interchangeably in this application.

As used herein, “sequence identity” is determined by using thestand-alone executable BLAST engine program for blasting two sequences(bl2seq), which can be retrieved from the National Center forBiotechnology Information (NCBI) ftp site, using the default parameters(Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which isincorporated herein by reference in its entirety). To use the term“homologus to” is synonymous with a measured “sequence identity.” Insome embodiments, if an embodiment comprises a nucleic acid sequence oramino acid sequence with a percent sequence identity the term refers toa disclosed nucleic acid sequence or amino acid sequence possessing ahomology to a disclosed sequence over its entire length.

The term “subject” is used throughout the specification to describe ananimal from which a sample of bodily fluid is taken. In some embodiment,the animal is a human. For diagnosis of those conditions which arespecific for a specific subject, such as a human being, the term“patient” may be interchangeably used. In some instances in thedescription of the present disclosure, the term “patient” will refer tohuman patients suffering from a particular disease or disorder. In someembodiments, the subject may be a human suspected of having or beingidentified as at risk to develop an aminoacidopathy. In someembodiments, the subject may be diagnosed as having at least oneaminoacidopathy. In some embodiments, the subject is suspected of havingor has been diagnosed with hyperammonemia. In some embodiments, thesubject may be a human suspected of having or being identified as atrisk to develop hyperammonemia. In some embodiments, the subject may bea mammal which functions as a source of the isolated sample of bodilyfluid. In some embodiments, the subject may be a non-human animal fromwhich a sample of bodily fluid is isolated or provided. The term“mammal” encompasses both humans and non-humans and includes but is notlimited to humans, non-human primates, canines, felines, murines,bovines, equines, and porcines.

As used herein, “conservative” amino acid substitutions may be definedas set out in Tables A, B, or C below. Metabolic enzymes include thoseamino acid sequences wherein conservative substitutions have beenintroduced by modification of polynucleotides encoding polypeptidesdisclosed herein. Amino acids can be classified according to physicalproperties and contribution to secondary and tertiary protein structure.A conservative substitution is recognized in the art as a substitutionof one amino acid for another amino acid that has similar properties.Exemplary conservative substitutions are set out in Table A.

TABLE A Conservative Substitutions I Side Chain Characteristics AminoAcid Aliphatic Non-polar G A P I L V F Polar - uncharged C S T M N QPolar - charged D E K R Aromatic H F W Y Other N Q D E

Alternately, conservative amino acids can be grouped as described inLehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY,N.Y. (1975), pp. 71-77) as set forth in Table B.

TABLE B Conservative Substitutions II Side Chain Characteristic AminoAcid Non-polar (hydrophobic) Aliphatic: A L I V P Aromatic: F W YSulfur-containing: M Borderline: G Y Uncharged-polar Hydroxyl: S T YAmides: N Q Sulfhydryl: C Borderline: G Y Positively Charged (Basic): KR H Negatively Charged (Acidic): D E

Alternately, exemplary conservative substitutions are set out in TableC.

TABLE C Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val Leu Ile Met Arg (R) Lys His Asn (N) Gln Asp (D)Glu Cys (C) Ser Thr Gln (Q) Asn Glu (E) Asp Gly (G) Ala Val Leu Pro His(H) Lys Arg Ile (I) Leu Val Met Ala Phe Leu (L) Ile Val Met Ala Phe Lys(K) Arg His Met (M) Leu Ile Val Ala Phe (F) Trp Tyr Ile Pro (P) Gly AlaVal Leu Ile Ser (S) Thr Thr (T) Ser Trp (W) Tyr Phe Ile Tyr (Y) Trp PheThr Ser Val (V) Ile Leu Met Ala

It should be understood that the polypeptides comprising polypeptidesequences associated with the extracellular matrix described herein areintended to include polypeptides bearing one or more insertions,deletions, or substitutions, or any combination thereof, of amino acidresidues as well as modifications other than insertions, deletions, orsubstitutions of amino acid residues.

As used herein, the term “prognosing” means determining the probablecourse and/or outcome of a disease.

As used herein, the term indophenol related compound—a small chemicalcompound that is a reaction product of an indophenol reaction. In someembodiment, it comprises at least one carbon atom in a 4, 5, 6-memberedring and emits a visible wavelength of light upon excitation of thesmall chemical compound by light emitted by from light source. In someembodiments, the small chemical compound is a product of the indophenolreaction and emits a wavelength of light visible to the human eye uponexcitation of the chemical compound by light emitted from a lightsource. In some embodiments, the small chemical compound emits awavelength from about 400 nm to about 600 nm when it is excited by lightfrom a light source. In some embodiments, the biosensor, device, and/orsystem comprises a light source and at least one diode and/orspectrophotometer, or other device capable of measuring the lightemitted by the indophenol or the indophenol related compound.

The term “vessel” as used herein is any chamber, indentation, container,receptacle, or space. In some embodiments, a vessel is a well capable ofholding no more than about 1,000, 900, 800, 700, 600, 500, 400, 300,200, 100, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μL ofsample. bodily fluid.

The term “membrane” means any monomer or polymer in a solid phase. Insome embodiments, the membrane comprises an ionomer. In someembodiments, the membrane is incapable of gas chromatography.

The terms “point of care” disclosed herein refer to a device, biosensor,system, test strip, or catridge, either individually or configured tofunction with one or more additional components, capable of analyzingthe presence, absence, or quantity of a reaction product, such asammonia, and/or a sample component, such as an amino acid, within a timeperiod no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or30 minutes. In some embodiments, the terms refer to a device, biosensor,system, test strip, or catridge, either individually or configured tofunction with one or more additional components, capable of analyzingthe presence, absence, or quantity of ammonia and/or an amino acidwithin a time period no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, or 30 minutes, or capable analyzing the presence, absence,or quantity of ammonia and/or an amino acid at or substantially near thepoint from which the sample was taken. For instance, in someembodiments, the sample may be taken from a subject suspected of orpreviously diagnosed with hyperammonemia or a hyperammonemia-relateddisorder. Without sending and analyzing the ammonia content of a sampleto a different location from the source of the sample, in someembodiments, the point of care device or biosensor or system is a pointof care device which is capable of detecting the presence, absence, orquantity of ammonia or ammonium ion in a sample.

The term “fluid exchange opening” means any space or void through whicha fluid may pass from one vessel to an adjacent vessel or another vesselin fluid commuinication with the one vessel.

The terms “individually comprise” in repsect to a claimed element orelements mean that only one claimed element comprises each of the listedelements and not in combination with any other element named.

The terms “a compound comprising a phenol substituent” means anymolecule comprising a phenyl group attached to a 4, 5, 6, ormore-membered atomic ring comprising at least one carbon atom.

The term “ionomer” as used herein refers to any polymer comprising anion. In some embodiments, the ionomer is a perflurinated ionomer. Insome embodiments, the ionomer comprises Formula I or a salt thereof.

Where X₁═F—O—CF₂—Y, F₂—SO₂, or F₂—CF₂—CO₂CH₃

X₂═CF₃, or, if X₁ is F₂, X₂ is null

Where Y═CF₂—SO₂F, CF₂—CF—SO₂F, or CF₃—CO₂CH₃In some embodiments, the ionomer comprises one or a combination of:

or a salt thereof, wherein n and m are any positive integer. In someembodiments, n and/or m are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. Insome embodiments, n and/or m are independently variable and any positiveinteger from about 1 to about 1000. In some embodiments, n and/or m areindependently variable and any positive integer from about 1 to about500.

The term “bodily fluid” means any sample taken from an animal includinga human, or non-human animal.

As used herein, the term “functional fragment” means any portion of adisclosed polypeptide that is of a sufficient length to retain at leastpartial biological function that is similar to or substantially similarto the function of the wild-type polypeptide upon which the fragment isbased. In some embodiments, a functional fragment of a polypeptideassociated with the function of a metabolic enzyme is a polypeptide thatcomprises at least 70%, 75%, 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity of any polypeptides disclosed herein and has sufficient lengthto retain at least partial binding affinity to one or a plurality ofsubstrates that bind to the polypeptide. In some embodiments, thefragment is a fragment of any polypeptide disclosed herein and has alength of at least about 10, about 20, about 30, about 40, about 50,about 60, about 70, about 80, about 90, or about 100 contiguous aminoacids. In some embodiments, the fragment is a fragment of anypolypeptide disclosed herein and has a length of at least about 50 aminoacids. In some embodiments, the fragment is a fragment of anypolypeptide disclosed herein and has a length of at least about 100amino acids. In some embodiments, the fragment is a fragment of anypolypeptide disclosed herein and has a length of at least about 150amino acids. In some embodiments, the fragment is a fragment of anypolypeptide disclosed herein and has a length of at least about 200amino acids. In some embodiments, the fragment is a fragment of anypolypeptide disclosed herein and has a length of at least about 250amino acids.

As used herein, the terms “polypeptide sequence associated with themetabolic enzyme” means any polypeptide or fragment thereof, modified orunmodified by any macromolecule (such as a sugar molecule ormacromolecule), that is a metabolic enzyme as diclosed herein or afunctional fragment thereof. In some embodiments the polypeptidesequence is is synthetic or recombinantly produced in any multicellularor unicellular organism. In some embodiments, a polypeptide sequenceassociated with the extracellular matrix is any polypeptide whichsequence comprises any of the polypeptides disclosed in Table 2. In someembodiments, a polypeptide sequence associated with the metabolic enzymeis any polypeptide sequence comprising any of the polypeptides disclosedin Table 2 or a sequence that shares 85,90,95, 96, 97, 98, or 99%sequence identity with the polypeptides disclosed in Table 2 or afunctional fragment thereof. In some embodiments, a polypeptide sequenceassociated with the metabolic enzyme consists of any of the polypeptidesdisclosed in Table 2 or a sequence that shares 85, 90, 95, 96, 97, 98,or 99% sequence identity with the polypeptides disclosed in Table 2.

The term “salt” refers to acidic salts formed with inorganic and/ororganic acids, as well as basic salts formed with inorganic and/ororganic bases. Examples of these acids and bases are well known to thoseof ordinary skill in the art. Such acid addition salts will normally bepharmaceutically acceptable although salts of non-pharmaceuticallyacceptable acids may be of utility in the preparation and purificationof the compound in question. Salts include those formed fromhydrochloric, hydrobromic, sulphuric, phosphoric, citric, tartaric,lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulphonic andbenzenesulphonic acids.

In some embodiments, the deivce, system, membrane, or vessel, maycomprise any of the disclosed reagents or formula disclosed herein orany salt. Salts may be formed by reacting the free base, or a salt,enantiomer or racemate thereof, with one or more equivalents of theappropriate acid. In some embodiments, salts of the present inventionrefer to salts of the disclosed reagents or formula disclosed hereinhaving at least one basic group or at least one basic radical. In someembodiments, salts of the present invention refer to salts of thedisclosed reagents or formula disclosed herein having a free aminogroup, a free guanidino group, a pyrazinyl radical, or a pyridyl radicalthat forms acid addition salts. In some embodiments, salts of thepresent invention refer to salts of the disclosed reagents or formuladisclosed herein that are acid addition salts of the subject compoundswith (for example) inorganic acids, such as hydrochloric acid, sulfuricacid or a phosphoric acid, or with suitable organic carboxylic orsulfonic acids, for example aliphatic mono- or di-carboxylic acids, suchas trifluoroacetic acid, acetic acid, propionic acid, glycolic acid,succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malicacid, tartaric acid, citric acid or oxalic acid, or amino acids such asarginine or lysine, aromatic carboxylic acids, such as benzoic acid,2-phenoxy-benzoic acid, 2-acetoxybenzoic acid, salicylic acid,4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such asmandelic acid or cinnamic acid, heteroaromatic carboxylic acids, such asnicotinic acid or isonicotinic acid, aliphatic sulfonic acids, such asmethane-, ethane- or 2-hydroxyethane-sulfonic acid, or aromatic sulfonicacids, for example benzene-, p-toluene- or naphthalene-2-sulfonic acid.When several basic groups are present mono- or poly-acid addition saltsmay be formed. The reaction may be carried out in a solvent or medium inwhich the salt is insoluble or in a solvent in which the salt issoluble, for example, water, dioxane, ethanol, tetrahydrofuran ordiethyl ether, or a mixture of solvents, which may be removed in vacuoor by freeze drying. The reaction may also be a metathetical process orit may be carried out on an ion exchange resin. Salts according to thepresent invention may be found in their anhydrous form or as in hydratedcrystalline form (i.e., complexed or crystallized with one or moremolecules of water).

As used herein, the term “antibody” refers to any immunoglobulin,whether natural or wholly or partially synthetically produced. In someembodiments, an antibody is a complex comprised of 4 full-lengthpolypeptide chains, each of which includes a variable region and aconstant region, e.g., substantially of the structure of an antibodyproduced in nature by a B cell. In some embodiments, an antibody is asingle chain. In some embodiments, an antibody is cameloid. In someembodiments, an antibody is an antibody fragment. In some embodiments,an antibody is chimeric. In some embodiments, an antibody isbi-specific. In some embodiments, an antibody is multi-specific. In someembodiments, an antibody is monoclonal. In some embodiments, an antibodyis polyclonal. In some embodiments, an antibody is conjugated (i.e.,antibodies conjugated or fused to other proteins, radiolabels,cytotoxins). In some embodiments, an antibody is a human antibody. Insome embodiments, an antibody is a mouse antibody. In some embodiments,an antibody is a rabbit antibody. In some embodiments, an antibody is arat antibody. In some embodiments, an antibody is a donkey antibody. Insome embodiments, the biosensor or system described herein comprises anantibody or plurality of antibodies.

Characteristic:

As is used herein, the term “characteristic” refers to any detectablefeature of a sample of bodily fluid that allows it to be distinguishedfrom a comparable sample of bodily fluid. In some embodiments, acharacteristic is an amount or identity of ammonia or ammonium ion inbodily fluid, in an environmental sample, or water sample. In someembodiments, a characteristic is an amount, sequence of, or modificationof a amino acid. In some embodiments a characteristic is an amount of acarbohydrate. In some embodiments, a characteristic is an amount of asmall molecule.

Comparable:

As is used herein, the term “comparable” is used to refer to twoentities that are sufficiently similar to permit comparison, butdiffering in at least one feature.

Metabolic Enzyme:

As is used herein, the term “metabolic enzyme” means an enzymeresponsible for catalysis of at least one step in the metabolic pathwayof one or more amino acids. In some embodiments, the metabolic enzyme isphenylalanine dehydrogenase, glutamate dehydrogenase, respectivefunctional fragments or a combination thereof or a fusion proteinthereof.

As used herein the terms “metabolic disease” is any one of a group ofdisorders caused by a defect in an enzymatic step in the metabolicpathway of one or more amino acids or in a protein mediator necessaryfor transport of certain amino acids into or out of cells. In someembodiments, the metabolic disease is chosen from: Argininemia (ARG,arginase deficiency) Argininosuccinate acidemia (ASA, argininosuccinase)Citrullinemia type I (CIT-I, argininosuccinate synthetase) Citrullinemiatype II (CIT-II, citrin deficiency) Defects of biopterin cofactorbiosynthesis (BIOPT-BS) Defects of biopterin cofactor regeneration(BIOPT-RG) Homocystinuria (HCY, cystathionine beta synthase)Hyperphenylalaninemia (H-PHE) Hypermethioninemia (MET) Maple syrup urinedisease (MSUD, branched-chain ketoacid dehydrogenase) Phenylketonuria(PKU, phenylalanine hydroxylase) Tyrosinemia type I (TYR-1,fumarylacetoacetate hydrolase), Tyrosinemia type II (TYR-II, tyrosineaminotransferase), and Tyrosinemia type III (TYR-III,hydroxyphenylpyruvate dioxygenase) where the parenthetical phrases aftereach disease state represent an abbreviation for the disease accompaniesby the enzyme that is generally defective in the subject suffering fromthe disease state.

Polypeptide:

The term “polypeptide”, as used herein, generally has its art-recognizedmeaning of a polymer of at least three amino acids. Those of ordinaryskill in the art will appreciate that the term “polypeptide” is intendedto be sufficiently general as to encompass not only polypeptides havingthe complete sequence recited herein, but also to encompass polypeptidesthat represent functional fragments (i.e., fragments retaining at leastone activity) of such complete polypeptides. Moreover, those of ordinaryskill in the art understand that protein sequences generally toleratesome substitution without destroying or significantly reducing activity.Thus, any polypeptide that retains activity and shares at least about30-40% overall sequence identity, often greater than about 50%, 60%,70%, 75%, 80%, or 85%, and further usually including at least one regionof much higher identity, often greater than 90% or even 95%, 96%, 97%,98%, or 99% in one or more highly conserved regions, usuallyencompassing at least 3-4 and often up to 20 or more amino acids, withanother polypeptide of the same class, is encompassed within therelevant term “polypeptide” as used herein.

As used herein, the term “threshold value” is the concentration ofammonia or ammonium ion or amino acid in a sample of bodily fluid thatindicates whether the amount of ammonia or ammonium ion or amino acid inthe sample is considered abnormally high or low resulting in a diagnosisor suspected diagnosis of a particular disorder, such as a metabolicdisease. For instance, in the case of a blood sample, known thresholdvalues for certain aminoacidopathies are indicated in Table 1 below:

TABLE 1 Aminoacidopathies and their associated amino acid markersdetectable in a sample Disorder Marker Abnormal Range ARG Arginine >100umol/L ASA Argininosuccinic acid  >4.0 umol/L ASA/Arg  >0.75 CIT-I andCIT-II Citrulline  >60 umol/L Cit/Tyr >1.0 Cit/Arg >6.0 HCY and METMethionine  >70 umol/L Met/Phe >1.2 MSUD Leucine >250 umol/L Valine >250umol/L Leu/Phe >4.0 Val/Phe >3.5 PKU, H-PHE Phenylalanine >130 umol/LBIOPT-BS and BIOPT-RG Phe/Tyr >2.0 TYR-I, TYR-II, and TYR-IIITyrosine >250 umol/LIn some embodiments, information about a threshold value or referencesample of bodily fluid is obtained prior to or simultaneously withinformation about an experimental sample of bodily fluid. In someembodiments, information about a reference cell or cell type ishistorical. In some embodiments, information about a threshold value orreference sample of bodily fluid is stored for example in acomputer-readable storage medium. In some embodiments, comparison of aparticular concentration value with a threshold value or referencesample of bodily fluid differentiates the concentration values ofammonia in an experimental sample of bodily fluid with the thresholdvalues thereby allowing a comparison that results in diagnosing asubject with one or more metabolic diseases or a change in severity ofone or more metabolic diseases.

Reference Electrode:

As will be understood from context, a reference electrode or controlelectrode is an electrically conductive support such as an electrodeplaced in a circuit with an at least one electrically conductive supportcomprising hydrogel and/or immobilized enzymes disclosed herein, topermit a relevant comparison of voltage difference between the referenceor control electrode and the at least one electrically conductivesupport comprising hydrogel and/or immobilized enzymes disclosed herein.

Sample:

As used herein, the term “sample” refers to a biological sample obtainedor derived from a source of interest, as described herein. In someembodiments, a source of interest comprises an organism, such as ananimal or human. In some embodiments, a biological sample comprisesbiological tissue or fluid. In some embodiments, a biological sample maybe or comprise bone marrow; blood; blood cells; ascites; tissue or fineneedle biopsy samples; cell-containing body fluids; free floatingnucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritonealfluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs;vaginal swabs; oral swabs; nasal swabs; washings or lavages such as aductal lavages or broncheoalveolar lavages; aspirates; scrapings; bonemarrow specimens; tissue biopsy specimens; surgical specimens; feces,other body fluids, secretions, and/or excretions; and/or cellstherefrom, etc. In some embodiments, a biological sample is or comprisesbodily fluid. In some embodiments, a sample is a “primary sample”obtained directly from a source of interest by any appropriate means.For example, in some embodiments, a primary biological sample isobtained by methods selected from the group consisting of biopsy (e.g.,fine needle aspiration or tissue biopsy), surgery, collection of bodyfluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, aswill be clear from context, the term “sample” refers to a preparationthat is obtained by processing (e.g., by removing one or more componentsof and/or by adding one or more agents to) a primary sample. Forexample, filtering using a semi-permeable membrane. Such a “processedsample” may comprise, for example nucleic acids or proteins extractedfrom a sample or obtained by subjecting a primary sample to techniquessuch as amplification or reverse transcription of mRNA, isolation and/orpurification of certain components, etc. in some embodiments, themethods disclosed herein do not comprise a processed sample.

In some embodiments, the system, test strip, device, biosensor, and/orcatridge comprises a concentration of any one or combination of thereagents disclosed on pages 78-84 of this disclosure.

TABLE 2 Enzyme Gene Sequence Accession Numbers PhenylalanineATGGAAATCTTCGAGGAAATCAAACGGCGGGGACACGAGCAAATTCTGTT AEW06037.1Dehydrogenase CAATTATGATCGGGCTTCCGGTTTGAAAGCAATTATCGCCATTCACAATAYP_005257709.1 CTACGTTGGGGCCGGCGTTGGGCGGGTGCCGAATGTTACCGTATCAAACGAEH47572.1 GAAGAGGCGGCCCTCGAGGATGCGCTGCGGTTGTCGGAAGGGATGACCTAYP_004587653.1 TAAAGCGGCCGCCGCCGGGCTCGATTTCGGCGGGGGCAAAACGGTGATTAYP_004581770.1 TCGGGGATCCGATGAAAGACAAGTCCGAGGCCCTGTTTCGTGCGCTCGGGAEH07849.1 CGTTTTATCGAGACCTTGAAAGGCCGTTACCTTACGGGAGAAGACGTAGG ACF96938.1AACCAACGAAGAAGATTTTGTCTGGGCTCGTCGGGAAACCCGTTATGTTG YP_007466124.1TCGGATTGCCGCCGGCTTATGGCGGGTCCGGCGATACGGGTGACAATACC EZP75760.1GCGCGCGGCGTCATTCAAGCGATGCGCGCCGCGTTGATGCACCGGTACGG AGT95551.1TTCGCCGGATCTCCAGGGCCGGCGGATTGCCGTCCAAGGGCTGGGCAAAG EWG09095.1TAGGCTATCATGTGGCGCGACGGGCCATCGAGGCCGGCGCTCGAGTGATT YP_008456272.1GCGGCCGATATCAATCCGCATGTAGTCGGCCGAGTGGCGTCCGCTTGGGG EME23486.1GATTGAAGCCACCGATCCGTGGGCTGTGGTGGAAACCCCCTGCGATATTT EJS99791.1TCGCCCCCTGTGCGTTGGGTAACGTCATTACGGAACGGACCGTGTCCGCC EIT85807.1CTCCAATGTCAGGTGGTGGCCGGTTCGGCCAACAATCAGCTGGCGGATGA AAA22646.1TCGACTGGCCGATGATTTAGCTGCCCGCGGCATTCTCTATGCGCCGGATT EDL64419.1TTATTGCGAATGCCGGCGGATTGATTCAGGTGGCGGATGAAATTCGGGGA EAR66050.1TATCATGAAGAACGGGTCCGTCATCAAATAGACGGGATTTATGACGTCCT BAA08816.1GCTCGAGATTTTTCGGAAGGCGGACGCCTCCGGCCGATCAACCGTGGCGGTTGCGGTAGACGAGGCGCGTCGCCGTTTGGACACCATTCAGGCCATCCAC CGCCTGTACGGATCATAGPhenylalanine CTGCAGGTCAACGGATCATATTCTACACATATATAATGCACTCCAATTGAAAA34179.2 Ammonia-LyaseCATAATACATAACGTGACATATGATACATTTATTAATATTAATTGTCACA ADR78835.1TTTACACTTCACATATTAAAATACTCTCGTATGAATGCAATTTGAAACAT AAA99500.1ATTTTAAATTAATTGATTGATATATATTGAACAAAACCTATTCACAAAGA AAC18871.1AACTTTCTTCTATTTCTCACTTATTTCTGCTAGTGTCTTTCCTATTCAAA AAC18870.1GCCATCATTTCCATCAACCTTCACAATACCATGTTTAAAAAGTCATTAAA AAA33805.1AATCAATTTTTTAAATAGAAAAAAACAAGAAGATGGAAATCACTTGGTTG AIC66437.1GTACTATATATTTAGTTGTTAAGTTTGACTCATACCGTGTATTGACCAAT AGY49231.1ATAAATAAAATCTTATTTCAAATAAATTCAAAAGTTCAATAAATATATAT AEW43005.1TCGTTCATAACTTATAATAAAATTGATTATACATAGTCCTCCCCCATTCA AFP24940.1CTTTTACTGATCAATTATTTCTAAAATATATTATTACTTTTACTTGTTAT AER58180.1TTTTAATAAATTAAGAAAATATAATACTCCCTTCGTTTTTAAAAAAATAC ADD12041.1CTAGTTTGACTTGAAACGGAGTTTAATAAAAGAAAGAAGACTTGTTAATC AEE81750.1TTGTGATTCTAAATTAAAGTTATGTCAAATGTACCAAAATGTCCTTTAAT AAP59440.1CTTGTGGTCTTAAACATGTCACATGAAAAATTAAAGTGTTTCCAAAAAAA AAP59439.1GAAAGGGGTCAATGTCATTCTTTTTTAAACAGACTAAAAAAGAAATAAAC AAP59438.1TCATTCTTTTTGAAACGGAGAGAGTAATTTTTTCCACGTTTTACTCATTA ACG80829.1ATATTAAATATTATTCTCTAGATCATCCTATAAGATCTAATAGTGGACAT ACG80828.1CAATTAATACCTATGTCACTTATTATTATTTTAATAATTGTATCAAGTCA ACG56648.1AATAATAACAAGTAAAAATGGAGTACCTACTATTAATCTTCAACAACCAC ACG56647.1AATTTACTAGTTTTTTCCTAGCAACCCCCTCTCACATATTTCACCATTTACTGGTTTTTTCCTAGCAACCCCCTCTCACATATTTTGTTTACCAACCATCATTTGTTCCTCTATATACTCACCACATGATAGATACATATATATACCACAACCAAAACAAAAGGTTTTATAAGTTCACAACATTTTTTATATACATACAAATAAACTCTAACCATTTTCTCTTCACTAAAAATTTCTTCATTACAAATCTAACAATTTACTTGATCCAATGGCACCATCAATTGCACAAAATGGACATATTAATGGAGAAGTAGCTATGGATTTGTGCAAGAAATCAATCAATGATCCATTGAATTGGGAAATGGCTGCTGATTCTTTAAGAGGCAGCCATTTGGATGAAGTGAAAAAGATGGTGGATGAATTTAGAAAGCCAATTGTGAAACTTGGGGGTGAAACTTTGTCAGTTGCACAAGTTGCATCCATTGCAAATGTTGATGACAAAAGTAATGGGGTTAAAGTGGAACTTTCTGAAAGTGCAAGGGCTGGTGTGAAAGCTAGTAGTGATTGGGTTATGGATAGTATGAGTAAAGGTACAGATAGTTATGGTGTTACTGCTGGATTTGGAGCAACATCTCATAGAAGAACAAAAAATGGTGGTGCTCTTCAAAAAGAACTTATTAGGTAAACAAACTATTTTTTTTCGTTATATATACTAACAATGTAAAGAATTTAATATTTTTTTGTTATATATACTAACAATGTAAAAAATTTAATATTTTTTTGTTATATATACTAACAATGTAAGAATTTAATATTTTTTTGTTATACATAGCTTATCGACTACTTAAGTGCTCCATTGATAAAGATTTTTTTTTGTTTTTACGCGAAGGGGATTCGGATGAATTCAGTTAAAATGTGATCTTAATGAATTATGATATTTTTTTGTAGGTTCTTGAATGCTGGAGTTTTTGGTAATGGAATAGAATCATTTCACACATTGCCACATTCAGCAACAAGGGCAGCTATGCTTGTTAGGATCAACACTCTGCTTCAAGGCTACTCTGGCATTAGATTTGAGATCTTGGAAGCAATCACTAAGTTGATCAATAGCAACATCACCCCGTGTTTGCCTCTCCGTGGCACGATCACTGCCTCGGGTGATCTCGTCCCTTTGTCCTATATTGCTGGTTTGCTCACTGGCAGACCTAATTCCAAGGCTGTTGGACCCAATGGTGAGAAACTTAATGCTGAGGAAGCTTTCTGCGTGGCTGGTATTAGTGGTGGATTTTTCGAGTTGCAGCCTAAGGAAGGACTTGCACTTGTGAATGGCACAGCAGTTGGTTCTGCTATGGCATCAATAGTCCTGTTTGAGTCCAATATCTTTGCTGTTATGTCTGAAGTTTTATCAGCGATTTTTACTGAAGTGATGAACGGAAAGCCCGAATTCACTGACTATTTGACACACAAGTTGAAGCATCACCCTGGTCAGATTGAGGCTGCTGCTATTATGGAACACATTTTGGATGGAAGCTCTTATGTGAAGGTAGCTCAGAAGCTCCATGAAATGGATCCTCTTCAAAAACCAAAGCAAGATCGTTATGCTCTCCGAACATCTCCACAATGGCTTGGACCTCAGATTGAAGTCATTCGTGCTGCAACTAAGATGATCGAGAGGGAGATTAACTCAGTGAACGACAATCCATTGATCGATGTTTCAAGAAACAAGGCCTTACATGGTGGCAACTTCCAAGGAACCCCTATTGGTGTCTCCATGGATAATACAAGATTGGCCCTTGCATCAATTGGTAAATTGATGTTTGCCCAATTCTCAGAGCTTGTCAACGACTATTACAACAACGGGTTGCCATCTAATCTGACAGCAGGAAGGAATCCAAGCTTGGACTATGGTTTCAAGGGCGCTGAAATCGCGATGGCTTCTTACTGCTCGGAACTTCAATTCTTGGCAAATCCAGTGACTAACCATGTCTAAAGTGCTGAGCAACACAACCAAGATGTGAATTCCTTGGGCTTAATTTCAGCCAGGAAAACAGCTAAGGCTGTTGATATCTTGAAGATAATGTCATCAACCTATCTCGTGGCTCTTTGCCAAGCTATTGACTTACGACATTTGGAGGAAAACTTGAAGAGTGTTGTCAAGAACACAGTTAGCCAAGTAGCTAAGAGAACTTTGACAATGGGTGCTAATGGTGAACTTCATCCAGCAAGATTCAGCGAAAAAGAATTGCTTCGAGTCGTGGATAGAGAATACTTGTTTGCCTATGCTGATGATCCCTGCAGCTCCAACTACCCTTTGATGCAGAAGCTGAGACAAGTCCTTGTTGATCAAGCAATGAAGAATGGTGAAAGTGAGAAGAATGTCAACAGCTCAATCTTCCAAAAGATTGGAGCTTTCGAGGACGAATTAATCGCTGTGTTGCCTAAAGAAGTTGAGAGTGTAAGAGCTGTTTTTGAAAGTGGCAACCCTTTAATTCGTAACAGGATCACAGAATGCAGATCATATCCATTGTACAGGTTGGTGAGAGAAGAACTTGGAACAGAATTGTTGACGGGTGAAAAAGTTCGATCACCTGGTGAGGAGATTGATAAGTGTTTACAGCAATATGTAATGGACAGATTATTGATCCATTGTTGGAGTGTCTGAAGAGCTGGAATGGTGCTCCTCTTCCAATCTGCTAAATGTGTTATTCTTTCAAGTTCTTTTTTTGTACCTTTTAGTGAATTACTAGAATTATAATGATGTTATGAACTTATATTAAAAAAAAATATTTTTGACTATAAAATTTAGTTTTGTTATTGAAATTAAAGGCTCAATCTGTGTTCTTTCCTTCTGTTATCTGAATATTATAAGAATTCAAGTAATCTTTTAGCTTTGTGAACAT GATGACATGCTTTCTTHistidine ATGATCACGCTTACCCCCGGCCACCTGACCCTCCCGCAACTGCGCCAGAT BAG44062.1Ammonia-Lyase CGCGCGCGAGCCCGTGCAGCTGACGCTGGATCCGGCCAGCTTCGCGAAGAYP_005225923.1 TCGACGCGGGCGCGAAGGCCGTGTCCGACATCGCCGCGAAGGGCGAGCCGCDF52938.1 GCGTACGGCATCAACACGGGCTTCGGTCGTCTGGCCAGCACGCATATCCC ABR76232.1GCACGATCAGCTCGAATTGCTGCAGAAGAACCTCGTGCTGTCGCATGCAG AAL19728.1TCGGTGTCGGCGAGCCGATGGCGCGTTCGTCGGTGCGTCTGCTGATCGCG AEW60321.1CTGAAGCTGTCGAGCCTCGGCCGCGGCCATTCGGGCATTCGCCGCGAAGT AEW51583.1GATGGACGCGCTGATCAAGCTGTTCAACGCCGACGTGCTGCCGCTGATTC ABQ54772.1CGGTGAAGGGCTCGGTCGGCGCATCGGGCGACCTCGCGCCGCTCGCGCAC AAX64695.1ATGTCGGCCGTGCTGCTCGGCGTCGGCGAAGTGTTCATTCGCGGCGAGCG AAU27462.1CGCGAGCGCGGTGGACGGGTTGCGCGTCGCGGGCCTCGCGCCGCTGACGC WP_021000087.1TGCAGGCGAAGGAAGGCCTCGCGCTGCTGAACGGTACGCAGGCGTCGACG YP_005185682.1GCGCTCGCGCTCGACAACCTGTTCGCGTACGAAGACCTGTACCGCACGGC YP_001250118.1GCTCGTCGCCGGCGCGCTGTCGGTCGATGCGGCGGCCGGCTCGGTGAAGC EFC47317.1CGTTCGACGCGCGCATCCACGAACTGCGCGGCCATCGCGGCCAGATCGAT AAH89809.1GCGGCGGCCGCGTATCGCGAGCTGCTCGAAGGCTCGGCGATCAACCTCTC BAH62483.1GCATCGCGACTGCGGCAAGGTGCAGGATCCGTACAGCCTGCGCTGCCAGC XP_002680061.1CGCAGGTGATGGGCGCGTGCCTGGACCAGATGCGTCATGCGGCCGACGTG AAO73411.1CTGCTCGTCGAGGCGAACGCGGTATCGGACAACCCGCTGATCTTCCCGGA CAI79696.1TACCGGCGAAGTGCTGTCGGGCGGCAATTTCCATGCGGAGCCCGTCGCGT CAI79696.1TCGCGGCCGACAACCTCGCGCTCGCGGCTGCGGAAATCGGCGCGCTGGCCGAGCGCCGCATCGCGCTGCTGATCGACGCGACGCTGTCGGGCCTGCCGCCGTTCCTCGTGAAGGATGGCGGCGTGAACTCGGGCTTCATGATTGCGCACGTGACGGCAGCTGCGCTCGCATCGGAGAACAAGACGCTCGCGCATCCGGCGTCGGTCGATTCGCTGCCGACCTCGGCGAACCAGGAAGACCACGTGTCGATGGCGACGTTCGCGGCACGCAAGCTGGCCGACATCGCCGACAAGACGAAGCACATCCTCGCGATCGAACTGCTCGCGGCCGCGCAGGGCGTCGATCTGCGCGAGAACGAGACGAGCCCGAAGCTCGCGGAAGTGATGAAGACGATTCGCAGCAAGGTCGCGCATTACGAGCTCGACCACTACTTTGCGCCGGACATCGCCGTGATCGCGAAGCTCGTCGTCGAGCGCGCGTTCGCGAAGCACTGCCCGTTC GCCTTCGCATCGGAGCAGTAATyrosine GTGACGCAGGTCGTGGAACGTCAGGCTGATCGGCTCAGCAGCAGGGAGTAYP_007039999.1 Ammonia-LyaseCCTGGCCCGGGTCGTGCGCAGCGCCGGGTGGGACGCCGGTCTCACCTCGT Q8GMG0.1GCACCGACGAGGAGATCGTCCGGATGGGCGCGAGCGCGCGCACCATCGAG WP_015103237.1GAGTACCTGAAGTCCGACAAGCCCATCTACGGCCTGACGCAGGGCTTCGG CCH33126.1TCCGCTGGTGCTGTTCGACGCCGACTCGGAGCTGGAGCAGGGCGGCTCGC AGZ04575.1TGATCTCGCACCTGGGCACCGGCCAGGGCGCGCCACTGGCCCCGGAGGTG GAK34477.1TCGCGGCTGATCCTCTGGCTGCGCATCCAGAACATGCGCAAGGGGTACTC AIG26365.1GGCGGTCTCGCCGGTGTTCTGGCAGAAGCTCGCCGACCTGTGGAACAAGG WP_030814263.1GGTTCACCCCGGCGATCCCCCGGCACGGCACGGTCAGCGCGAGCGGCGAC WP_030592622.1CTGCAACCGCTGGCGCACGCCGCGCTCGCCTTCACCGGTGTCGGCGAGGC WP_030583802.1GTGGACCCGGGACGCCGACGGCCGGTGGTCCACCGTGCCGGCCGTGGACG WP_030225885.1CGCTCGCCGCGCTGGGGGCGGAGCCGTTCGACTGGCCGGTGCGCGAGGCG WP_030107056.1CTGGCGTTCGTCAACGGGACCGGCGCGAGCCTCGCGGTGGCTGTGCTCAA WP_010261615.1CCACCGGTCCGCCCTGCGGCTGGTCCGCGCCTGCGCCGTGCTCTCCGCGC WP_009065811.1GGCTGGCGACCCTGCTGGGGGCCAATCCCGAGCACTACGACGTGGGGCAC WP_029043904.1GGTGTCGCGCGCGGCCAGGTCGGTCAGCTGACCGCGGCGGAGTGGATCCG WP_029027607.1GCAGGGGCTGCCCCGGGGCATGGTGCGCGACGGCAGCCGCCCGCTCCAGG WP_029025670.1AGCCGTACAGCCTGCGGTGCGCGCCGCAGGTGCTCGGCGCGGTGCTCGAC WP_029023988.1CAGCTCGACGGCGCGGGCGACGTGCTGGCGCGGGAGGTCGACGGCTGCCA WP_029020280.1GGACAACCCGATCACCTACGAGGGCGAGCTGCTGCACGGCGGCAACTTCC WP_028673581.1ACGCCATGCCGGTGGGTTTCGCCTCCGACCAGATCGGGTTGGCCATGCACATGGCCGCCTACCTGGCCGAGCGCCAGCTGGGTCTGCTGGTCAGCCCGGTGACCAACGGCGACCTGCCGCCCATGCTCACCCCGCGCGCCGGGCGCGGTGCCGGGCTGGCCGGGGTGCAGATCAGCGCGACCTCGTTCGTCTCGCGGATCCGGCAGCTGGTGTTCCCCGCCTCGCTGACCACCCTGCCGACCAACGGCTGGAACCAGGACCACGTGCCGATGGCGCTCAACGGGGCGAACTCGGTGTTCGAGGCGTTGGAGCTCGGCTGGCTGACGGTCGGGTCGCTGGCGGTGGGCGTCGCGCAGCTCGCGGCCATGACCGGCCACGCCGCGGAGGGCGTCTGGGCGGAGCTGGCCGGGATCTGCCCGCCGCTGGACGCCGACCGCCCGCTGGGCGCCGAGGTGCGCGCCGCGCGCGACCTGCTGTCCGCGCACGCGGACCAACTGCTCGTCGACGAGGCAGACGGGAAGGATTTCGGATGA GlutamateATGTCAGCAAAGCAAGTCTCGAAAGATGAAGAAAAAGAAGCTCTTAACTT P39633.3Dehydrogenase ATTTCTGTCTACCCAAACAATCATTAAGGAAGCCCTTCGGAAGCTGGGTTKEG08275.1 ATCCGGGAGATATGTATGAACTCATGAAAGAGCCGCAGAGAATGCTCACTNP_001233850.1 GTCCGCATTCCGGTCAAAATGGACAATGGGAGCGTCAAAGTGTTCACAGGNP_001268039.1 CTACCGGTCACAGCACAATGATGCTGTCGGTCCGACAAAGGGGGGCGTTCAEW04907.1 GCTTCCATCCAGAAGTTAATGAAGAGGAAGTAAAGGCATTATCCATTTGGYP_007161255.1 ATGACGCTCAAATGCGGGATTGCCAATCTTCCTTACGGCGGCGGGAAGGGYP_005256579.1 CGGTATTATTTGTGATCCGCGGACAATGTCATTTGGAGAACTGGAAAGGCYP_004932652.1 TGAGCAGGGGGTATGTCCGTGCCATCAGCCAGATCGTCGGTCCGACAAAGYP_004442444.1 GATATTCCAGCTCCCGATGTGTACACCAATTCGCAGATTATGGCGTGGATYP_004412348.1 GATGGATGAGTACAGCCGGCTGCGGGAATTCGATTCTCCGGGCTTTATTAYP_004410986.1 CAGGTAAACCGCTTGTTTTGGGAGGATCGCAAGGACGGGAAACAGCGACGYP_004372731.1 GCACAGGGCGTCACGATTTGTATTGAAGAGGCGGTGAAGAAAAAAGGGATYP_004367667.1 CAAGCTGCAAAACGCGCGCATCATCATACAGGGCTTTGGAAACGCGGGTAYP_004366366.1 GCTTCCTGGCCAAATTCATGCACGATGCGGGCGCGAAGGTGATCGGGATTYP_004343968.1 TCTGATGCCAATGGCGGGCTCTACAACCCAGACGGCCTTGATATCCCTTAYP_004343356.1 TTTGCTCGATAAACGGGACAGCTTTGGTATGGTCACCAATTTATTTACTGYP)004261766.1 ACGTCATCACAAATGAGGAGCTGCTTGAAAAGGATTGCGATATTTTAGTGYP_004270382.1 CCTGCCGCGATCTCCAATCAAATCACAGCCAAAAACGCACATAACATTCAYP_004099961.1 GGCGTCAATCGTCGTTGAAGCGGCGAACGGCCCGACAACCATTGATGCCAYP_003967811.1 CTAAGATCCTGAATGAAAGAGGCGTGCTGCTTGTGCCGGATATCCTAGCGAGTGCCGGCGGCGTCACGGTTTCTTATTTTGAATGGGTGCAAAACAACCAAGGATATTATTGGTCGGAAGAAGAGGTTGCAGAAAAACTGAGAAGCGTCATGGTCAGCTCGTTCGAAACAATTTATCAAACAGCGGCAACACATAAAGTGGATATGCGTTTGGCGGCTTACATGACGGGCATCAGAAAATCGGCAGAAGCATCGCGTTTCCGCGGATGGGTCTAA GlutamateATGTCCATCAAAGACGCTGTAAAACTGATTGAAGAAAGCGAAGCCCGCTT CBX22311.1Ammonia-Lyase TGTCGATTTGCGCTTTACCGATACCAAAGGCAAGCAGCACCACTTTACCGTGCCTGCGCGCATCGTGTTGGAAGACCCCGAAGAGTGGTTCGAAAACGGACAGGCGTTTGACGGTTCGTCCATCGGCGGCTGGAAAGGCATTCAGGCTTCCGATATGCAGCTTCGCCCCGATCCCGCCACGGCGTTTATCGATCCTTTTTATGATGATGTTACCGTCGTCATTACCTGCGACGTTATCGATCCCGCCGACGGTCAGGGTTACGACCGCGACCCGCGCTCCATCGCACGCCGCGCCGAAGCCTATTTGAAATCTTCCGGTATCGGCGACACGGCATACTTCGGTCCCGAACCCGAGTTTTTCGTCTTCGACGGCGTAGAATTTGAAACCGATATGCACAAAACCCGTTACGAAATCACGTCCGAAAGCGGCGCATGGGCCAGCGGCCTGCATATGGACGGTCAAAACACCGGCCACCGCCCTGCCGTCAAAGGCGGTTACGCGCCCGTCGCGCCGATTGACTGCGGTCAGGATTTGCGTTCCGCGATGGTAAACATTTTGGAAGGACTCGGCATCGAAGTCGAAGTGCACCACAGCGAAGTCGGTACCGGCAGCCAAATGGAAATCGGCACGCGCTTCGCCACCTTGGTCAAACGCGCCGACCAAACCCAAGACATGAAATATGTGATTCAAAATGTCGCCCACAACTTCGGCAAAACCGCCACCTTCATGCCCAAACCCATTATGGGCGACAACGGCAGCGGTATGCACGTTCACCAATCCATCTGGAAGACGGTCAAAACCTGTTCGCAGGCGACGGCTATGCCGGCTTGAGCGACACCGCGCTCTACTACATCGGCGGCATCATCAAACACGCCAAAGCCCTGAACGCGATTACCAATCCGTCCACCAACTCCTACAAACGCCTTGTGCCGCACTTTGAAGCGCCGACCAAACTGGCATATTCCGCCAAAAACCGTTCCGCTTCCATCCGTATTCCGTCTGTGAACAGCAGCAAGGCGCGCCGCATCGAAGCGCGTTTCCCCGACCCGACCGCCAACCCGTACTTGGCGTTCGCTGCCCTGCTGATGGCGGGTTTGGACCGGCATTCAAAACAAAATCCATCCGGGCGATCCTGCCGATAAAAATCTCTACGACCTGCCGCCGGAAGAAGACGCGCTCGTCCCGACCGTTTGCGCTTCTTTAGAAGAAGCCCTCGCCGCGCTCAAAGCCGACCACGAATTCCTCTTACGCGGCGGCGTGTTCAGCAAAGACTGGATCGACAGCTACATCGCCTTTAAAGAGGAAGATGTCCGCCGCATCCGTATGGCGCCGCATCCGCTGGAATTTGA AATGTATTACAGCCTGTAAThreonine AGGAGGTGTTTTAATAATGAAAGGTTTTGCAATGCTCAGTATCGGTAAAG NP_622353.1Dehydrogenase TCGGTTGGATTGAAAAAGAAAAGCCTACTCCCGGCCCTTTTGACGCTATTEPX86072.1 GTAAGACCTCTAGCTGTGGCCCCTTGCACTTCGGACGTTCATACCGTTTT AFT82159.1TGAAGGTGCTATTGGCGAAAGACATAACATGATACTCGGTCACGAAGCTG YP_006796158.1TAGGTGAAGTAGTTGAAGTAGGTAGTGAGGTAAAAGATTTTAAACCTGGT EJZ15419.1GATCGCGTTGTGGTACCAGCTATTACCCCTGATTGGCGAACCTCTGAAGT YP_001727630.1GCAAAGAGGATATCACCAACACTCTGGTGGAATGCTGGCAGGCTGGAAAT ACA82186.1TTTCGAATATAAAAGATGGTGTTTTTGGTGAATTTTTTCATGTGAACGAT AGZ44086.1GCTGATATGAATTTAGCACATCTGCCTAAGGAAATTCCATTGGAAGCTGC AEB44998.1AGTTATGATTCCCGATATGATGACTACTGGCTTTCACGGAGCCGAACTGG YP_008737139.1CAGATATAGAATTAGGTGCGACGGTAGCGGTTTTGGGTATTGGCCCAGTA EPX87740.1GGTCTTATGGCAGTCGCTGGTGCCAAATTGCGGGGTGCTGGAAGGATTAT YP_004405598.1CGCAGTAGGCAGTAGACCAGTTTGTGTAGATGCTGCAAAATACTATGGAG BAN60779.1CTACTGATATTGTAAACTATAAAGATGGTCCTATCGACAGTCAGATTATG EPE39095.1GATTTAACGGAAGGCAAAGGTGTTGATGCTGCCATCATCGCTGGAGGAAA EPC57128.1TGTTGACATCATGGCTACAGCAGTTAAGATTGTTAAACCTGGTGGCACCA EME23086.1TCGCTAATGTAAATTACTTTGGCGAAGGAGATGTTTTGCCTGTTCCTCGT ACI75705.1CTTGAATGGGGTTGCGGCATGGCTCATAAAACTATAAAAGGCGGGCTATG ACI75704.1CCCCGGTGGACGTCTAAGAATGGAAAGACTGATTGACCTTGTTGTTTATA ACI75703.1AGCGTGTCGATCCTTCTAAGCTCGTCACTCACGTTTTCCGGGGATTTGAC ACI75702.1AATATTGAAAAAGCCTTTATGTTGATGAAAGACAAACCAAAAGACCTAATCAAACCTGTTGTAATATTAGCATAA ThreonineATGGCTGACTCGCAACCCCTGTCCGGTACCCCGGAAGGTGCCGAATATTT EGP22802.1Ammonia-Lyase AAGAGCGGTGCTGCGCGCGCCGGTCTACGAAGCGGCGCAGGTCACGCCGCAIL15845.1 TACAGAAAATGGAAAAACTGTCGTCGCGTCTCGATAACGTGATTCTGGTG KFJ14411.1AAGCGCGAAGATCGCCAGCCAGTTCATAGCTTTAAGTTGCGCGGCGCATA B22317CGCCATGATGGCGGGCCTGACGGAAGAACAAAAAGCACACGGCGTGATTA ESE06785.1CCGCTTCTGCAGGTAACCACGCGCAGGGCGTCGCGTTTTCTTCCGCACGG ESD87895.1TTAGGCGTGAAGGCGCTGATCGTCATGCCAACCGCCACCGCCGATATCAA ESD77040.1AGTTGATGCGGTGCGCGGCTTTGGCGGCGAAGTGCTGCTTCACGGCGCAA ESD56952.1ATTTCGATGAAGCGAAAGCGAAAGCGATCGAACTGTCACAGCAGCAGGGT ESD26867.1TTCACCTGGGTACCGCCGTTCGATCATCCGATGGTGATCGCCGGGCAAGG ESD18649.1CACGCTGGCGCTGGAACTGCTCCAGCAGGACGCCCATCTCGACCGCGTAT ESC98561.1TTGTACCGGTCGGCGGCGGCGGTCTGGCAGCGGGTGTGGCGGTGCTGATC ESA95751.1AAACAACTGATGCCGCAAATCAAAGTAATCGCCGTGGAAGCGGAAGATTC ESA86931.1CGCCTGCCTGAAAGCGGCGCTGGATGCGGGTCATCCCGTTGATCTGCCCC ESA78951.1GCGTGGGGCTGTTTGCTGAAGGCGTCGCGGTAAAACGCATCGGCGATGAA ESA72735.1ACCTTCCGTTTGTGCCAGGAGTATCTTGACGACATCATCACCGTCGATAG ESA67809.1CGATGCCATCTGTGCGGCGATGAAAGATCTGTTCGAAGATGTGCGCGCGG ERL21545.1TGGCGGAACCTTCCGGCGCGCTGGCGCTGGCGGGGATGAAAAAATACATC ERK40933.1GCCCAGCACAACATTCGCGGTGAACGGCTGGCGCATATTCTTTCCGGTGC ERJ97484.1TAACGTGAACTTTCACGGTCTGCGCTACGTCTCGGAACGCTGCGAACTGG ERH28800.1GCGAACAGCGTGAAGCGTTGTTGGCGGTGACCATTCCGGAAGAAAAAGGCAGCTTCCTCAAATTCTGCCAACTGCTTGGCGGGCGTTCGGTCACCGAGTTCAACTACCGTTTTGCCGATGCCAAAAACGCCTGCATCTTTGTCGGCGTGCGCTTAAGCCGTGGCCTCGAAGAGCGCAAAGAAATTTTGCAGATGCTCAACGACGGTGGCTACGCGTGGTTGATCTCTCCGACGACGAAATGGCGAAGCTGCATGTGCGCTATATGGTTGGCGGGCGTCCATCGCATCCGTTGCAGGAACGCCTATACAGCTTCGAATTCCCGGAATCACCGGGCGCGCTGCTGCGCTTCCTCAACACGCTGGGTACGCACTGGAACATCTCGCTGTTCCATTATCGCAGCCACGGTACCGACTACGGGCGCGTACTGGCGGCGTTCGAGCTTGGCGATCATGAACCGGATTTTGAAACCCGGTTGAATGAACTGGGCTACGATTGCCACGACGAAACCAATAACCCGGCGTTCAGGTTCTTTTTGGCGGGTTAG SerineATGAGCGGTACCATCCTCATCACCGGCGCCACGTCCGGCTTCGGACAGGC ADY67207.1Dehydrogenase CACGGCGCGGCGTTTCGTCAAGGAAGGCTGGAAGGTCATCGGCACAGGTCYP_004444298.1 GGCGGGCGGAACGGCTGGAGGCGCTGGCGCAAGAACTCGCTCCGCCTTTCEAZ63492.1 ACGGCGCTGCCTTCGATGTTACCGACGAAGATGCCACTAGAAAGGCACTTXP_001387515.1 GCGGCTTTGCCGGAAGGTTTCCGGGACATCGATATTCTCGTCAACAATGCBAB07807.1 GGGGCTTGCGCTCGGCACCGCACCTGCACCGCAGGTGCCGCTGAAAGACT EMS96834.1GGCAGACCATGGTGAACACCAACATCACCGGTCTTTTGAACATCACCCAC EKJ96295.1CATCTTTTGCCCACGTTGATCGACCGCAAGGGCATTGTCATCAACCTTTC EHJ96027.1CTCGGTAGCTGCGCACTGGCCCTATGCGGGCGGCAATGTCTATGCCGGAA EHH03760.1CGAAAGCCTTCCTGCGGCAATTCTCGCTCGGTCTGCGCTCCGACCTGCAT WP_028707025.1GGCAAGGGCGTGCGCGTCACCTCGATCGAACCGGGCATGTGCGAAACGGA NP_356536.1ATTCACGCTTGTTCGCACCGGCGGCAATCAGGATGCCTCGGACAATCTTT AEQ50417.1ACAAGGGCGTCAATCCGATCACGGCCGAGGATATCGCCAATACGATCCAT AAK89321.1TGGGTCGCCTCGCAGCCCAAACATATCAACATCAACAGCCTCGAACTCAT YP_004898167.1GCCGGTCAACCAGTCCTTTGCCGGTTTCCAAGTGCATCGGGAAAGTTGA YP_064393.1WP_003522480.1 EGP55658.1 EGL63994.1 KFC62486.1 WP_031354348.1 SerineATGATGACCAAAAACGAAATCCAAAAGTGGGTAAAGGAATTCCCGCTGCT KFL14920.1Ammonia-Lyase TGAAACGATCATGGCGGCCGAAGAGGTATTTTGGCGCAATCCAAAATATCAIF56070.1 ACGCGTTTGCGCAAGCTATTCGAACGATTCCTTTACGCGAACGCGATGTC KFI03369.1AAGGAGGCCGAAGAGCGATTGCGCCGCTTTGCCCCCTACATCGCGAAAGT KFH36969.1GTTTCCCGAGACGCGAACGGCCCACGGTATCATCGAATCCCCTTTAGTGC KFH35774.1GGATTCCGAACATGAAACAGCGTTTGGAAAAGATGTTTCAGACCAACATC KFH56112.1GAGGGGGATCTGTTGCTAAAATGCGACAGCCATCTTCCCATCTCCGGATC WP_031409141.1GATCAAGGCGAGAGGGGGAATCTACGAGGTTCTGAAACATGCGGAAGAAC KFC30598.1TCGCTCTGGCAAACCATATGATCACCATGGGGGATGACTATGCGGTCATG KEZ84476.1GCCAGCGAAGAATTCCGGCAGTTCTTTTCCCGCTATTCGCTTGTCGTTGG KEY95863.1TTCGACGGGAAATTTAGGCTTGAGTATCGGCATCATCGGGGCGCAGCTTG KER46054.1GGTTCCGCGTTACCGTTCATATGTCAGCCGATGCGAAACAATGGAAAAAA WP_030024949.1GACTTGTTGCGAAGCAAAGGGGTTGCGGTCATCGAACATCTCACCGACTA KEK24273.1CAACAAGGTGGTGGAAGAGGCGCGAAGACAGTCCGCCGAGGATCCAACGT KEK22892.1CGTATTTTATCGATGATGAGAACTCGATCCATCTGTTTTTAGGCTATGCG KEK18491.1GTGGCGGCGTTTCGGCTGAAAAAGCAATTAGAGGACATGAACATCACGGT KEK12402.1TGATGAAAACCACCCGCTCTTTGTATATCTTCCTTGCGGCGTCGGCGGCG WP_029761212.1GTCCGGGCGGGGTGACGTTTGGGCTGAAGCTCGTGTACGGCGATCATGTC WP_029758174.1CATTGCTTTTTCGCTGAGCCGACGCATTCGCCTTGCATGTTGCTCGGCCT WP_029714078.1GATGACGGGACAGCACGACCGCGTGTCGGTGCAAGATTTTGGCCTCGACA WP_029598316.1ATAAGACCGAAGCGGACGGGCTAGCGGTGGGGCGGCCGTCAAGGTTGGTGGGGAACATGCTTGAGAACGTCATCAGCGGCGTCTATACGGTGGACGATGCGACGCTTTACCGCTTGCTCGCGGCGATGGTGGAAACGGAGGAAATCTATTTAGAGCCGTCCGCCTTGGCGGGGGTGGCGGGGCCTGTTCGGCTGTTTCGTGATTTGGCGGGGCAAACGTACGTAGAGGCAAACGGTTTGAAAGAAAAGATGAAAAACGCCGTCCATATTGGCTGGGCGACAGGCGGAAGCATGGTGCTAAAGGATGTGATGGAGGCCTATTATCGGGAAGGCGTGCGCATCGAAACGATGACAGGGAACGGTTTTTCTGAAGGACGATAA LeucineATGCTGATGTTCGAAGAAATCCAGGCGCGCGGCCACGAGAGCGTCACGCT YP_004169785.1Dehydrogenase GCTGCACCACGCCCCCAGCGGCCTGCGCGCCGTGCTCGCCGTGCACTCCAADV66120.1 CCGTGCTCGGCCCTGCCATTGCCGGCTGCCGCCTGATGCCCTGCACCGAG ADY26991.1GAACGCGCCGTGCGCGACGCCCTCGCCCTCAGCGAGTCCGTCACGCTCAA AEW05136.1GGCCGCCCTCGCGGGCCTGAACTACGGCGGGGGCGCGTGCGTCATGCTCC YP_005256808.1CCCCGGAAGGCGGCGACATCGACGGGCACGCCCGCGAGGCGCTGTTCCGC YP_004256608.1GCGCTCGGCCGGCAGATCCGTTACCGCGGTGGCCGCGTCATCCTCACCGA YP_004346245.1GGACGTCGGCGTGACCGGCCGCGACATCGCCTTCGCCGCGCAGGAAACCG AEA45407.1ACAGCACCATGGGCATGCACACCGACACGCCCACCGTCACCGCGTACGGC YP_004101992.1GTGTACCGCGGCATCAAGGCCGCCGCGCGCGCCTACCTCGGCGGCGAGAG YP_004101991.1CATGCGCGGCGTGCGCGTCGCGCTGCTCGGCGCGGGCGCAGTCGGGCGCA YP_003825932.1CCCTCGCGCAGCACCTGCACCGCGAGGGCGCGCGCCTCACCGTCGCAGAC ADU51265.1CTGATGTCTGAGCGCGCGCAGGCCCTCGCGGACGACCTCGGCGAGCGCGT ADU51264.1CACCGTCGTGAGCGCCGCTGACATCTTCGACGTGCCGTGCGACGTATTCG ADU08309.1CGCCGTGCGCGTTCGGGCACAGCATCAAAAGCGCCGACGTGCCCCGCTTG AFY88585.1CAGTGCCGGGTGATCGCCGGCAGCGAACACCACCCGCTCAGCCACAACGG YO_004054007.1CGAGACGCTCGTGCGCGAAGCGGGCATCACATACATCCCGGACTTCGCCA YP_007092454.1TCAACAGCGCCGGCCTGATGAGCGCCGCGCAGAACCTCAGCATCGAAACG YP_003825216.1GCGGCGGAACGCGTGTACGAGAGCGTCGCGCAGATCTGCGCGACCGCGCA ADR21899.1GAAGTACGAGAAGCCGCCGCACGTCGTCGCCCGTAAACTCGCGCTGCGCC ADL07593.1GCATCGAACTGATCGGCTCCATCAGCGGCCAGTACGCCGGCCAGTAA AsparteTCATGTGCCAACACGTATGTTATCACTTAAAATTTTTAGTAAAGTGACTG ADP76847.1Dehydrogenase CTGAATATGCTGCCAAAACACTTGTTTTTGGATTTAATTCACACACAGTGYP_004003609.1 TTTTTTGTTATAGATTTAAACTCTCCAAAATCTCCTTTAACATGGACTTCADE37476.1 ATGGATATTGTGTTCAACTTCAGGATCTGCAATTATCTTTACATCCGCAT AEH60264.1CTATTCCAGAGGCTAGACTTAATGCCGCAGCAACGTTAATATTCACTGGA AEH50568.1AATTTTTTAACAGCTTCTGAGGATTTCCCTTTAAACACGACCTCCTTTTT YP_004615483.1TTTGGTCTTAACACCTAACGAAGTAGGTGATTTTCTCGTTATAAGTTTTA YP_004659664.1TTTCTTTTATCTTACCTAAGGATGCGGCTTTTACACCATCTAAACCAATT YP_003543121.1ATTGCACCGGAAGGTATGTATATATTAGCTCCTGATTCTCTAGATTCCTT YP_003895891.1TATCAATCTTCTTCTAACTTTCTCATCTAATAGTGCACCCACACTCATAA ADN37453.1TCAAAACATCTATACCTCTACTAATTATATTGGGCACAATTTCTTTTACT ADV47603.1GCCTCTTGAGAAGCAGATTCAATTATCAAATCAACTCCATTGAACATTTC YP_004163101.1TTCTACCTTTTTTACGGCAGTGCCATTTGTTAAATTTGCTAGCTTCTTAG ADY50896.1CTTTTCTAAAATTTCTGTCATAAAAATATTTTAATTTTATTTTTTTGATA ABX33598.1TCTTGTTTTAAGACAAGGTTAACTATTGTATTTGCAATTGCACCACATCC YP_004272718.1TATAATCCCACATCTCAT ADN60949.1 ACL18032.1 ACL16745.1 ADD08173.1 AspartateATGTCCTCGCCTGCATCATCGCGCATCGAAAAAGACCTGCTTGGTGTTCT ELS44542.1Ammonia-Lyase CGAAGTACCTGCCAACGCGTATTACGGCATCCAGACCCTGCGAGCGGTGAEXL32019.1 ACAACTTTCACCTCTCCGGCGTGCCGCTTTCGCACTACCCGAAACTGGTA EPF69098.1GTCGCGCTGGCCATGGTCAAGCAGGCGGCAGCGGATGCAAACCATCAGCT EDZ32290.1CGGACACCTCAATGACGCCAAGCATGCGGCGATCAGCGAGGCCTGTGCCC ACC77466.1GCCTGATCCGCGGCGACTTCCACGATCAGTTCGTGGTCGACATGATCCAG ETO09916.1GGCGGCGCTGGCACGTCGACCAACATGAATGCCAACGAAGTCATCGCCAA ETN58394.1CATCGCTCTGGAAACCATGGGTTTCGAGAAAGGCGCATACAAACACCTGC AGZ94384.1ACCCCAACAACGATGTCAACATGGCGCAGTCGACCAACGACGCCTACCCC EGU12843.1ACGGCGATCCGCTTGGGTCTGCTGCTGGGTCACGACGCTCTGCTCGCCAG AGQ54567.1CCTTTCCAGCCTGATTCAGGCCTTCGCCGCCAAGGGCGAAGAATTCAACC BAN21048.1ATGTGCTGAAGATGGGCCGCACCCAGTTGCAGGACGCCGTTCCAATGACC ELU36465.1CTGGGTCAGGAATTCCGCGCCTTCGCCACCACCCTGACAGAAGACCTGAA ELU36464.1CCGCCTGCGCAGCCTGGCGCCAGAGCTGTTGACCGAAGTGAACCTCGGCG EDS31003.1GAACCGCCATCGGCACCGGCATCAACGCCGACCCTGGCTATCAGAAGCTG BAM20634.1GCAGTCGATCGTCTGGCACTCATCAGCGGCCAGCCTCTGGTGCCAGCAGC ACO48312.1CGACCTGATCGAAGCGACCTCCGACATGGGCGCCTTCGTGTTGTTCTCGG XP_001828833.2GCATGCTCAAGCGTACTGCGGTCAAGCTGTCGAAAATCTGCAACGACCTG EAU92840.2CGCCTGCTGTCCAGCGGCCCACGCACCGGCATCAACGAAATCAACCTGCC XP_001849880.1GGCACGTCAGCCAGGCAGCTCGATCATGCCCGGCAAGGTCAACCCGGTGA XP_001658988.1TCCCGGAAGCGGTCAATCAGGTTGCCTTCGAAATCATCGGCAACGACCTGTCGCTGACCATGGCAGCCGAAGGAGGACAATTGCAGCTCAACGTGATGGAGCCGCTGATCGCCTACAAGATCTTCGACTCGATCCGCCTGCTGCAGCGCGCCATGGACATGCTGCGCGAGCACTGCATCGTCGGCATCACAGCCAACGAACAGCGCTGCCGCGAGCTGGTCGAGCATTCGATCGGTCTGGTCACCGCCCTGAACCCTTACATCGGTTACGAGAACTCCACCCGTATCGCCCGCATCGCGCTGGAAACCGGCCGCGGCGTGCTGGAACTGGTGCGTGAGGAAGGTCTGCTCGACGACGCCATGCTCGACGACATCCTGCGCCCGGAAAACATGATCGCTCCGCGTCTGGCCCCCTTGAAGGCCTGA ValineTCAGCGACCGCGGGCCTCGGCCATCCGCTGCTCGGCGATCCGGTCGGCCG YP_007932652.1Dehydrogenase CCGCGGCGGGCGGAATGCCGTCCGCCTTCGCACGTGCGAATATTTCCAGCAGK78767.1 GTGGTGTCGAAGATCTTCGTCGCCTTCGCCTTGCACCGGTCGAAGTCGAANP_628270.1 CCCGTGCAGCTCGTCGGCGACCTGGATCACGCCGCCGGCGTTGACCACATYP_001973234.1 AGTCGGGTGCGTAGAGGACCGACCGGTCGGCCAGGTCCTTCTCGACACCCAIJ14557.1 GGGTGGGCCAGCTGGTTGTTGGCCGCGCCGCACACCACCTTCGCCGTGAGYP_007523209.1 CACCGGAACGGTCGCGTCGTTGAGCGCGCCGCCGAGCGCGCAGGGCGCGTWP_015659426.1 AGATGTCGAGACCCTCGGTGCGGATCAGCGTCTCGGTGTCCGCCACCACGCCK29082.1 GTGACCTCGGGGTGCAGATCGGTGATCCGGCGCACCGACTCCTCGCGTAC CAR62534.1GTCGGTGATCACGACCTCGGCCCCGTCGGAGAGCAGGTGCTCGACGAGGT AGT93561.1GGTGGCCCACCTTGCCGACCCCGGCGACGCCGACCTTGCGGCCGCGCAGC AEK45617.1GTCGGGTCGCCCCACAGGTGCTGGGCCGAGGCCCGCATGCCCTGGAAGAC ADI08852.1ACCGAACGCGGTGAGGACGGAGGAGTCGCCGGCGCCGCCGTTCTCGGGGG YP_008454282.1AGCGGCCGGTGGTCCAGCGGCACTCCCTGGCGACGACGTCCATGTCGGCG ESQ05180.1ACGTAGGTGCCGACATCGCAGGCGGTGACGTACCGGCCGCCGAGCGAGGC ESP98677.1GACGAACCGGCCGTAGGCCAGGAGGAGTTCCTCCGTCTTGATCTTCTCCG YP_005535074.1GGTCGCCGATGATGACGGCCTTGCCGCCACCGTGGTCGAGTCCGGCCAGG YP_004963983.1GCGTTCTTGTACGACATCCCGCGCGACAGGTTCAGCGCGTCGGCGACGGC EOD63988.1CTCGGCCTCGGTCGCGTACGGGTAGAAGCGGGTGCCGCCGAGGCCGGGGC EME98953.1CCAGGGCGGTGGAGTGGAGGGCGATGACGGCCTTGAGGCCGGTGGCACGG EME52779.1TCCTGGCAGATCACGACTTGCTCGTGACCCCCCTGATCCGAGTGGAACAGGGTGTGCAGGACGCCGTTAGTCACATCGGTCAC GlycineCTAGTTGTAAAAGTCGAGGGAGGCGCAACTGCACATGAGGTGACGATCTC KEG12217.1Dehydrogenase CGTAAACCCCGTCAATGCGACCCACAGTCGGCCAGTACTTTTCAACGTACADH66904.1 GAGTAAGGATAAGGGAATGCCGCCAAACGCCGATCATATGGTTTGTCCCAYP_003679410.1 TTTATCATCGGTGACACATCTTGCCGTGTGTGGTGCATTCTTCAAAACATYP_003507491.1 TGTTATCCACTGGTTGTTCACCTTTTTCAATGGCGGCAATCTCACCTCGAADN74845.1 ATGGAAATTAGTGCATCTGCCAAGCGATCCAACTCCCGCTTGGGTTCTGA ADD28471.1TTCGGTGGGTTCAATCATTAAAGTCCCGGGTACAGGAAACGCCAGTGTTG YP_004445203.1GCGAGTGAATTCCGTAGTCCATCAACCGTTTGGCCACGTCCTCCGCCTCA YP_003911919.1ATATGAGCTGTCTTCTTGAACCGTCGAAGATCAACGATAAACTCATGAGC ADQ81869.1GCAGTAGTTTTCTCCACCCAGGAAAAGAATCGTATAATGGTTCTCTAGGC AEH88507.1GCTTCTTCAAGTAGTTTGCATTCAAAACGGCGTACTCTGTACAAGTTTTG YP_007138219.1AGCCCGTGTGATCCAAGCATTAACATCAACATGTACGATATCGGAAGAAT YP_004612601.1TGATGCTGATCCGTACGCTGATTGTGAGACTTGGCCGAATGGCTGTGAAC YP_004170318.1CGCCAACTTTTTGGTTGAAAACAGAATTTGGCAAAAAGGGGGCCAGATGT YP_004163559.1TGACGGACAGCTATAGGGCCCATTCCGGGGCCGCCACCACCATGGGGAAT YP_004045375.1TGAAAACGTCTTGTGGAGATTAATGTGGCACACGTCGCCACCGATATATC YP_004787190.1CAGGGCCTGTATAGCCAACCATGGCGTTAAGATTTGCCCCATCAATGTAG YP_001742361.1CATTGTCCACCGTAGTAGTGCGCCATTGATGTAATGGATAAAATATCCTT YP_007067896.1GTCAAACAAGCCATACGTACTTGGATATGTTATCATGATACACGACAACT YP_007100788.1CCTTTGCGTGTTTTTGGCAAGATTTCTCCAGGTCATTGATATCAACCCTG YP_004773043.1CCGTTAGACAAGCATTTCACCAAGACAATATTCATTCCTGCCAATGTTGCCGAAGCTGGATTCGTACCATGCGCACTCTCTGGAATCAAACAGACGTTGCGGTGTCCTTCCTTCATTGATAGATGGTACGCACGAATAACACGAAGCCCAGCGTATTCACCTTGGGCGCCACTATTAGGCTGAAGCGATACCGCATCCAGACCGGTAATTTCCCTTAACTTTTGCTCAAGATCTAGACACAACGCACTGTACCCTCGCACTTGGTCCACTGGGGCAAGGGGATGCACATTGGTGAATTCTGGCCAAGAGAGTGGTAACATAGCAGCGGCAGGGTTAAGCTTCATGGTGCAAGATCCCAACGGGACGCAACCATGCGTAAGGCCGTAATCCTTTCGTTGTAGACGATGAATATAGCGCATCAGTTCACTTTCACTCTTGTACTTTTGAAACGTTGAGTGTTTCAGGAAATCAGACTTCCGCACCAGATCCAACGGTAGTACCGATTTCTGATCGGCTATTTTGGAAAGGGCTGCGACGACGGGAAGCTTCAACCCTGCAGCCTCCAAAAGTGACACAATGTGTCCATCCGTTGTTGCCTCATCCACAGAAATGGAGACAGTCCCATTACTGTAATCAACAAAAACATTAATACCCTTCTCAACACATCGTGTCTTGTAATCCTCCGCTGTAATGCCTTTTAGGTTCACAGTAACAGTGTCGAAAAATGCACTGTTTACCACAGAGTGTCCTACTGATTCCATACCAACAGCGAGCACTTTCGCCTTGCCGTGTATCTCATTGGCAATCTCATTTAGACCATCTGGACCATGGTAGGCGGCATAAAACCCACTCACGTTGGCCAATAACGCTTGTGCAGTACAGATATTTGATGTGGCGCGCTCACGCTTAATATGTTGTTCACGTGTCTGCAGCGCCATGCGTATGGATGGCTCTCCGGCAGAATCCTTACTGACGCCGATCACACGTCCCGGCATCAACCTCTTAAACTGCTCCTTGACAGCAAAGAACGCGGCGTGAGGACCTCCATATCCTAGTGGAACACCAAAACGCTGGGAGGATCCCACAACCACATCTGCATTCATTTCACCAGGTGGCTTGACAAGAACACAAGCCATCAAGTCGGTCCCACAGCAACTAATGACACCGTGCTTCTTTGCATTCTCGAACAGTGGTGAGAAGTCATGAAGCATGCCCATCGCATCTGGTGTTTGTACAAGGATACCAAACAAGGAACTGTCAGTCCAGTCAATCAGATTCGTGTCGCCCACGACGACGTTTATCTTGAGCGGTTCGGCTCTTGTCTTAACCATCTCAATGCAGGATGGAAAAACAGTTTTGATACGAAGAACGTATTCCGCTTTCGTTGACCATGCTGAAAAGCAAGATGCATCGCCTCGGATGATGTCTGTCGCTTGGTCAAGAAGAGATGCATTTGCCACATCCATCTTTGTCAAATCCATAACCATGGTTTGGAAATTCAAAAGGGACTCCAGACGTCCTTGTGCAATCTCAGCTTGGTATGGTGTGTAGGGTGTGTACCATCCAGGATTTTCAATGACGTTGCGAAGTATGACAGGAGGAGTAATGGACTCGTAGTACCCCTGACCAATCATGCTTTTTAGTACCTTGTTTCGCGCACCAAGAGAGCGACGAGTGCGAGAGCATCCATCTCACTCATAGCCGCCACCTCCGTCAAGGGTGGGCGTACAATATCCCCTGGAATAGCAGCCGTCATCAAATCAGAGACTCTCTTTTCCAACCGTTCGAAGCATCGACATTGTCTCAGCCGTTGTTGGACCAATATGGCGGTTAATATAGCTGTCCGTGGCAGTCCATCGAACAAATGTCACGCATGGCAAAGAGCCACGAAACAAACGAC GGTACAT AlanineATGATCATTGGCCTGCCGAAAGAGATCAAAGTTAAGGAAAACCGCGTGGC YP_004171395.1Dehydrogenase ACTCACGCCCGGGGGCGTCGCCAGCCTCGTGCGCCGCGGCCACACCGTCAADV67730.1 TCGTGGAACGCAGCGCCGGCGTGGGCAGCGGCATCCAGGACACCGAGTAC ADV25885.1GAGCAGGCCGGCGCGCAGCTCGGCAGCGCCGCCGAGGCGTGGGCCGCGCA ADV48359.1GATGGTCGTGAAGGTCAAGGAGCCCATCAAGAGCGAATACGGGTACCTCC AFZ35417.1GCCCGGACCTGCTGCTGTTCACGTACCTGCACCTCGCTGCGGACCAGCCC AFZ05172.1CTCACGGACGCCCTGCTGAGCGCCGGCACGACCGCCGTTGCGTACGAGAC AEW05285.1GGTGCAGCTCGACGACCGCAGCCTGCCGCTGCTCACGCCCATGAGTGAGG AEW04533.1TCGCGGGCCGCCTGAGCGTGCAGGCCGGCGCGTACCACCTGCAAAAGCCC AEM70054.1ATCGGCGGGCGCGGCGTGCTGCTCGGCGGCGTGCCGGGCGTGCAGGCGGG YP_005256957.1CCACGTCGTCGTGATTGGCGGCGGCGTCGTCGGCACGAACGCCGCGAAAA YP_005256205.1TGGCCATGGGCCTCGGCGCGAAGGTCACGGTGCTGGACGTGAACCACGGG YP_004450492.1CGCCTCTCGTACCTCGACGACGTGTTCTTCGGGAAGCTCACCACCATGAT YP_004368103.1GAGCAACGAGGCGAACATCCGCTCCATCCTGCCCGAAGCGGACCTCGTGA YP_004340432.1TCGGCGGCGTGCTGATCCCCGGGGCGAAGGCGCCGCACCTTGTCACGCGC YP_004261609.1GACATGCTGGCGACCATGCAGGAAGGCAGCGTCATCGTCGACGTGGCGGT YP_004255502.1GGACCAGGGCGGATGCGTGGAGACCATTCACGCGACGACGCACGACGATC YP_004163857.1CCACGTACATCGTGGACGGCGTGATCCACTACGGCGTGGCGAACATGCCG YP_004787486.1GGCGCGGTGCCGCGCACCAGCACGTTCGCGCTCACGAACCAGACCATTGG YP_007132437.1GTACGTGCTGCAGCTCGCGGACAAGGGCGTGGAGGCACTCAGCGCCAGCA YP_007113588.1AGCCGCTGCTGCGTGGCCTGAACACCATCGGCGGGAAGCTGACGTACGCGGGCGTCGCGGAAGCGTTCGGCCTGACGTACACCGCGCCTGAAGTGGCGCT GGCGTAA ProlineATGGAGCCCACTATGAGCCAATTCGAACAGCTGTACCGCCAGGTGGCCCT ADY26965.1Dehydrogenase CAGTGTCGCCGGCAACCCGGTCGTGGAAAAAGTCTTGAGCAAGCAGGGCTADI14966.1 GGGCGCTGGCGCAGCGTTTTGTATCGGGCGAGACGGCGCAGGACGCCATCYP_004437470.1 AAGGCCATCAAGCGGCTGGAAGCCCAGGGCATCTCCGGCAACCTCGACCTYP_004368974.1 GCTGGGCGAGTTCGTGAACACCCCGGAACCCGCCAATGCCAACACCGAGAYP_004345744.1 TGATTCTGGCGACCATTGACCAGGTGCACGCGGCGGGCCTCACGCCCTACYP_004340684.1 AACAGCGTGAAAATGTCGGCGCTGGGCCAAGGGCAGACCGCGCCGGACGGYP_004256582.1 CCAGGACCTCGGCTACGTCAACACCCGCCGCGTCGTGGAGCGGGCCAAGCYP_004170680.1 GCTACGGCGGCTTCGTCAATCTGGACATGGAAGACCACACCCGCGTGGACYP_003705539.1 TCGACTCTGCAGATTTTCCGCCGCCTGGTCAAGGAGTTCGGCCACCAGCAAEA44906.1 TGTGGGAACGGTGTTGCAGGCCTACCTGCACCGCTCGGAAGACGACCGCC AEB12864.1GCAGCCTGGACGACCTGCGCCCCAACCTCCGCATGGTGAAGGGCGCCTAC ADV67015.1CTGGAGCCCGCCTCCGTCGCCCTGCAGAGCAAAACCGACATTGACGCCGC AEE14339.1CTACCGCCGCCTGGTCTACGAGCACCTCAAGGCCGGCAACTACTGCAACG AEA34625.1TGGCCACCCACGACCACCACATCATCTACGACGTGATGCACTTTGCGCTG EFH82753.1GCCCACGGCATCCCTAAGGACCAGTTCGAATTCCAGCTGCTGTACGGCAT NP_868270.1CCGCGAGGACCTGCAGCGCGAATTGGCCGAGGCCGGCTACACGGTGCGCT ADQ16526.1CGTACATTCCTTTCGGCAAGGACTGGTACGGCTACTACTCGCGCCGCATC AEL26370.1GCCGAGCGCCCGCAGAACGTGATGTTCGTGCTGCGCGGCCTGCTGTAA AFK04422.1 AEM71761.1Lysine ATGAAAAACATTGTGGTTATCGGCGCGGGCAATATCGGTTCGGCAATCGC BAH80102.1Dehydrogenase CTGGATGCTGGCCGCATCAGGCGATTATCGCATCACGGTTGCCGATCGTTAAV93559.1 CAGCCGATCAGCTGGCCAATGTGCCGGCGCATGAACGGGTCGACATCGTCYP_165503.1 GACATTACCGACCGTCCCGCTCTGGAAGCACTGCTAAAAGGCAAATTCGCAIA01810.1 CGTGCTCTCCGCCGCCCCCACCGAATTCCACCTGACGGCGGGTATTGCCG AIA03878.1AAGCGGCCGTTGCCGTCGGCACGCATTATCTCGATCTCACCGAAGACGTG AIA03881.1GAATCCACCCGCAAGGTCAAGGCGCTGGCGGAAACGGCCGAAACCGCGCT AIA00889.1CATTCCGCAATGCGGCCTCGCCCCCGGCTTCATCTCCATCGTCGCTGCCG EXU92064.1ATCTCGCCGTCAAGTTCGACAAGCTGGACAGCGTGCGCATGCGCGTCGGC EOT00338.1GCTCTGCCGCAATATCCGTCCAATGCGCTCAACTACAACCTCACCTGGAG NP_882461.1TACCGACGGGCTGATCAACGAATATATCGAGCCCTGCGAAGGATTCGTCG EIJ80893.1AAGGCCGCCTCACCGCCGTTCCGGCCCTTGAGGAGCGCGAGGAGTTCTCG AIA06975.1CTCGATGGCATCACCTACGAGGCGTTCAACACCTCGGGCGGTCTCGGTAC AIA05885.1GCTTTGCGCGACGCTGGAAGGCAAGGTGCGGACCATGAACTACCGCACTA EXU88317.1TCCGTTATCCCGGCCATGTGGCGATCATGAAGGCGCTTTTGAACGACCTC EOT04629.1AACCTGCGCAACCGCCGCGATGTGCTGAAGGACCTGTTCGAAAACGCCCT AIA07859.1GCCCGGCACCATGCAGGATGTGGTCATCGTCTTCGTCACCGTCTGCGGCA AIA04644.1CCCGCAACGGCCGCTTCCTGCAGGAAACCTATGCCAACAAGGTCTATGCC AIA04440.1GGCCCGGTTTCCGGCCGGATGATGAGCGCCATCCAGATCACTACCGCCGC AIA03522.1CGGCATCTGCACGGTTCTCGACCTGCTCGCGGAAGGCGCCCTGCCGCAGA AIA02686.1AGGGCTTCGTTCGACAGGAGGAAGTGGCGCTGCCGAAGTTCCTCGAAAACCGGTTTGGCCGGTATTATGGCTCGCATGAGCCGCTGGCGCGGGTTGGGTG A

The disclosure relates to an ammonia or ammonium ion biosensor formeasuring a total concentration of a ammonia in the blood. The ammoniabio sensor comprises a measuring electrode which include as components,a mediator and an enzyme, which selectively act on the plurality ofspecific amino acids each serving as a substrate, and a counterelectrode. In the amino-acid biosensor, the enzyme has a substrateaffinity to each of the plurality of specific amino acids. The enzyme isoperable to catalyze a reaction in each of the plurality of specificamino acids as a substrate so as to form a reaction product. Themediator is operable, during amino-acid concentration measurement, tocarry electrons between the reaction product and the measuringelectrode. Further, the amino-acid bio sensor is designed to apply avoltage between the measuring electrode and the counter electrode at ameasurement point in such a manner that, in an analytical curverepresenting a relationship between an applied voltage and a currentvalue in a specific concentration for each of the plurality of specificamino acids, the applied voltage is a voltage allowing the variety ofthe current values for the amino acids in the same concentration and atthe same applied voltage.

In some embodiments, the measuring electrode (at least a firstelectrode) further comprises a a hydrogel that comprises a coenzyme orreduction agent as a component. In some embodiments, the enzyme consistsof a dehydrogenase. Further, the reaction product consists of a reducedcoenzyme derived by reduction of the coenzyme, and the mediator isoperable, during the amino-acid concentration measurement, to carryelectrons from the reduced coenzyme to the measuring electrode.

In some embodiments, a biosensor or system disclosed herein is used inconjunction with one or a combination of the following:

1. a power source in electrical connection with the electrodes andcapable of supplying an electrical potential difference between theelectrodes sufficient to cause diffusion limited electro-oxidation ofthe reduced form of the mediator at the surface of the workingelectrode; and

2. at least one meter, (such as a spectrophoteomter, voltmeter and/oramperometer) in electrical connection with the electrodes and capable ofmeasuring the diffusion limited current produced by of the reduced formof the mediator with the above-stated electrical potential difference isapplied.

The meter will normally be adapted to apply an algorithm to the currentmeasurement, whereby an ammonia or ammonium ion concentration isprovided and visually displayed. Improvements in such power source,meter, and biosensor system are the subject of commonly assigned U.S.Pat. No. 4,963,814, issued Oct. 16, 1990; U.S. Pat. No. 4,999,632,issued Mar. 12, 1991; U.S. Pat. No. 4,999,582, issued Mar. 12, 1991;U.S. Pat. No. 5,243,516, issued Sep. 7, 1993; U.S. Pat. No. 5,352,351,issued Oct. 4, 1994; U.S. Pat. No. 5,366,609, issued Nov. 22, 1994;White et al., U.S. Pat. No. 5,405,511, issued Apr. 11, 1995; and Whiteet al., U.S. Pat. No. 5,438,271, issued Aug. 1, 1995, the disclosures ofwhich are hereby expressly incorporated by reference.

Ammonia or ammonium ion concetrations from a plaurality of samples maybe analyzed in parallel. For example, human and non-human body fluidssuch as whole blood, plasma, sera, lymph, bile, urine, semen,cerebrospinal fluid, spinal fluid, lacrimal fluid and stool specimens aswell as other biological fluids readily apparent to one skilled in theart may be measured. Fluid preparations of tissues from humans andnon-human animals can also be assayed, along with foods, water samples,fermentation products and environmental substances, which potentiallycontain environmental contaminants. In some embodiments, human serum isassayed with the disclosed biosensor. In some embodiments, the biosensorcomprises or is configured to assay whole blood.

After reaction is complete, a power source (e.g., a battery) applies apotential difference between electrodes. When the potential differenceis applied, the amount of oxidized form of the mediator at the auxiliaryelectrode and the potential difference must be sufficient to causediffusion-limited electro-oxidation of the reduced form of theat leastone mediator at the surface of the working electrode. In someembodiments, the working electrode comprises a hydrogel disclosedherein. A current measuring meter (not shown) measures thediffusion-limited current generated by the oxidation of the reduced formof the mediator at the surface of the working electrode. The measuredcurrent may be accurately correlated to the concentration of ammonia orammonium ion and/or one or more amino acids in sample when the followingrequirements are satisfied:

1. The rate of the indophenol reaction based upon the concentration ofindophenol reagents is governed by the rate of diffusion of the ammoniafrom the sample in a first vessel to the second vessel comrpsing asurface of the working electrode.

To manufacture biosensor a roll of metallized film is fed through guiderolls into an ablation/washing and drying station. A laser systemcapable of ablating bottom plate element 14 is known to those ofordinary skill in the art. Non-limiting examples of which includeexcimer lasers, with the pattern of ablation controlled by mirrors,lenses, and masks. A non-limiting example of such a system is theLPX-300 or LPX-200 both commercially available from LPKF LaserElectronic GmbH, of Garbsen, Germany.

In the laser ablator, the metallic layer of the metallized film isablated in a pre-determined pattern, to form a ribbon of isolatedelectrode sets. The metallized film is further ablated, after theisolated electrode sets are formed to create recesses positionedadjacent the electrochemical area. The ribbon is then passed throughmore guide rolls, with a tension loop and through an optional inspectioncamera. The camera is used for quality control in order to check fordefects.

Reagent is compounded and applied in a liquid form to the center of theelectrochemical area at a dispensing and drying station. Reagentapplication techniques are well known to one of ordinary skill in theart as described in U.S. Pat. No. 5,762,770, the disclosure of which isexpressly incorporated herein by reference. It is appreciated thatreagent may be applied to array in a liquid or other form and dried orsemi-dried onto the center of the electrochemical area in accordancewith this disclosure.

In addition, a roll or top plate element material is fed into anassembly station along with a roll of spacer material. Liners on eitherside of the spacer material are removed in that station and the topplate element or surface scaffold is applied to one side of the spacermaterial to form a top plate element/spacer subassembly. The top plateelement/spacer subassembly is slit into the appropriate width for a rowof biosensors. Next, a new release liner is added to the side of thespacer material opposite the cover and the subassembly is wound into aroll.

The ribbon of the reagent-coated bottom plate element is unwound and fedinto a sensor assembly station along with the top plate element/spacersubassembly. The liner is removed from the spacer and the subassembly isplaced on bottom plate element to cover reagent. Next, the assembledmaterial is cut to form individual biosensors, which are sorted andpacked into vials, each closed with a stopper, to give packaged sensortest strips.

Although ablating recesses is described herein, it is appreciated thatthe method of forming recesses in bottom plate element is also notlimited. For example, the recesses may be formed by etching (e.g., usingphotoligographic methods) or otherwise removing a portion of the surfaceof top plate element. The nearest electrode edge is approximately about10 μm to about 500 μm from the recess, or about 100 μm to about 400 μmfrom the recess, or from about 200 μm to about 300 μm from the recess.Biosensors that are formed with recesses in accordance with thisdisclosure yield a reagent profile with generally uniform thickness ofchemistry. A generally uniform thickness of chemistry allows for moreaccurate sample analysis.

The processes and products described above include a disposablebiosensor, especially for use individually as a diagnostic device or incombination with other components such as a pump system orspectrophotometer configured to diagnose hyperammonemia, abnormalfunction, or abnormally high or low amounts of ammonia in a sample.

Variations on the Indophenol Reaction

The disclosure relates to contacting a sample with one or a plurality ofreagents in independently variable phases of dried, powdered or aqueousphases. The reaction has four major components: a compound comprising aphenyl group, a hypohalite, a catalyst and an alkali buffer. When thesereagents are exposed to ammonia, an indophenol compound is producedthat, when exposed to a light source at a particular wavelength, absorbsand/or emits a particular wavenlength of light. in some embodiments, anyof the methods disclosed herein make comprise a step of detecting thepresence, absence, or quantity of ammonia or ammonium ion by measuringthe absorbance of the contents of at least the first vessel or thesecond vessel.

Family of Phenols

Different compounds comprising a phenyl group can be used as long as thecompound comprises a 4, 5, or 6-membered ring with at least one carbonatom and a unsubstituted ‘para-position.’

Alkali Conditions

Any buffer capable of creating an alkali microenvironment for thereaction to take place with ammonia from a sample may be used. In someembodiments, a vessel comprising an alkali buffer with pH from about 8.5to about 13 can be used in the biosensor, test strip, or systemdisclosed herein. Any compound that can create these alkali conditionscan be used including sodium and potassium hydroxide, or sodium orpotassium acetate. In some embodiments, the alkali buffer is in apowdered form, lyophilized, or aqueous solution in a vessel locatedwithin the biosensor or kit disclosed herein.

Electrode

In some embodiments, the biosensor, system or test strip disclosedherein comprise one or more electrodes. In some embodiments, the one ormore electrodes transmit current variation generated by the reactionbetween the indophenol reagents and ammonia or ammonium ion from asample and/or transmit current variation generated by a battery sourceto the light source or other equipment necessary to provide a readout ofthe levels of ammonie in a sample, for instance, in the case of aspectrophotometer to measure absorbance of a reactant vessel in thebiosensor. In some embodiments, the electrodes comprise metal. In someembodiments, the electrodes comprise a carbon scaffold upon which ametal is deposited. In some embodiments, the electrodes comprise acarbon scaffold of carbon nanotubes.

Electrode structures which are suitable for the present disclosure andmethods for the production of such structures have already beensuggested in biosensor technology for other purposes. In this regard,reference is made to U.S. Pat. No. 6,645,359 and its content isincorporated herein by reference in its entirety. Electrodes orElectrically conductive tracks are created or isolated on first surface.Tracks represent the electrodes of biosensor. As used herein, the phrase“electrode set” is a set of at least two electrodes, for example 2 to200, or 3 to 20, electrodes. These electrodes may, for example, be aworking (or measuring) electrode and an auxiliary electrode. In someembodiments, tracks cooperate to form an interdigitated electrode arraypositioned within the periphery of recesses and leads that extend fromarray and between recesses toward end.

Tracks are constructed from electrically conductive materials.Non-limiting examples of electrically-conductive materials includealuminum, carbon (such as graphite), cobalt, copper, gallium, gold,indium, iridium, iron, lead, magnesium, mercury (as an amalgam), nickel,niobium, osmium, palladium, platinum, rhenium, rhodium, selenium,silicon (such as highly doped polycrystalline silicon), silver,tantalum, tin, titanium, tungsten, uranium, vanadium, zinc, zirconium,mixtures thereof, and alloys, oxides, or metallic compounds of theseelements. Preferably, tracks include gold, platinum, palladium, iridium,or alloys of these metals, since such noble metals and their alloys areunreactive in biological systems. In some embodiments, the track is aworking electrode made of silver and/or silver chloride, and track is anauxiliary electrode that is also made of silver and/or silver chlorideand is substantially the same size as the working electrode.

Tracks are isolated from the rest of the electrically conductive surfaceby laser ablation. Techniques for forming electrodes on a surface usinglaser ablation are known. Techniques for forming electrodes on a surfaceusing laser ablation are known. See, for example, U.S. patentapplication Ser. No. 09/411,940, filed Oct. 4, 1999, and entitled “LASERDEFINED FEATURES FOR PATTERNED LAMINATES AND ELECTRODE”, the disclosureof which is expressly incorporated herein by reference. Tracks arepreferably created by removing the electrically conductive material froman area extending around the electrodes. Therefore, tracks are isolatedfrom the rest of the electrically-conductive material on a surface by agap having a width of about 5 μm to about 500 μm, preferably the gap hasa width of about 100 μm to about 200 μm. Alternatively, it isappreciated that tracks may be created by laser ablation alone on bottomsubstrate. Further, tracks may be laminated, screen-printed, or formedby photolithography.

Multi-electrode arrangements are also possible in accordance with thisdisclosure. For example, it is contemplated that a biosensor may beformed that includes an additional electrically conductive track. In athree-electrode arrangement such as the arrangement depicted in FIG. 4,the first track is a working electrode, the second is a counterelectrode, and the third electrode is a reference electrode. It is alsoappreciated that an alternative three-electrode arrangement is possiblewhere tracks are working electrodes and a third electrode is provided asan auxiliary or reference electrode. It is appreciated that the numberof tracks, as well as the spacing between tracks in array may vary inaccordance with this disclosure and that a number of arrays may beformed as will be appreciated by one of skill in the art. in someembodiments, the electrodes are embedded on or attached to a solidsupport, such as a test strip comprising a plastic and/or papermaterial.

Micro-electrode arrays are structures generally having two electrodes ofvery small dimensions, typically with each electrode having a commonelement and electrode elements or micro-electrodes. If “interdigitated”the arrays are arranged in an alternating, finger-like fashion (See,e.g., U.S. Pat. No. 5,670,031). These are a sub-class ofmicro-electrodes in general. Interdigitated arrays of micro-electrodes,or IDAs, can exhibit desired performance characteristics; for example,due to their small dimensions, IDAs can exhibit excellent signal tonoise ratios.

Interdigitated arrays have been disposed on non-flexible substrates suchas silicon or glass substrates, using integrated circuitphotolithography methods. IDAs have been used on non-flexible substratesbecause IDAs have been considered to offer superior performanceproperties when used at very small dimensions, e.g., with featuredimensions in the 1-3 micrometer range. At such small dimensions, thesurface structure of a substrate (e.g., the flatness or roughness)becomes significant in the performance of the IDA. Because non-flexiblesubstrates, especially silicon, can be processed to an exceptionallysmooth, flat, surface, these have been used with IDAs. In someembodiments, the at least one electrode is a component of any IDAdisclosed herein.

Membrane

In some embodiments, the membrane positioned at a fluid exchange openingcomprises an ionomer. In some embodiments, the membrane comprises one ora combination of the following polymers:

wherein each of the variables p, q, r, s, t, u, v, w, x, y, and z areindependently variable and are 0 or any positive integers; and wherein Ris independently selected from an amine, hydroxy, hydroxyl, carbonyl, H,═O, —OH, —COOH, —N, —CH₃, —CH₂—X, halo, aryl, arylalkoxy, arylalkyl,alkynyl, alkenyl, alkylene, alkyl, akyl-halo, arylamido,alkylheterocycle, alkylamino, alkylguanidino, alkanol, alkylcarboxy,cyclo alkyl, heteroaryl, heteroarylalkyl, heteroarylalkoxy, orheterocyclyl; or any salt thereof.

In some embodiments, the R group is acidic or an electronegativesubstiuent. In some embodiments, the variables p, q, r, s, t, u, v, w,x, y, z are independently variable and are 0 or positive integers fromabout 1 to about 200. In some embodiments, the variables p, q, r, s, t,u, v, w, x, y, z are independently variable and are 0 or positiveintegers from about 10 to about 100. In some embodiments, the variablesp, q, r, s, t, u, v, w, x, y, z are independently variable and are 0 orpositive integers from about 10 to about 100 across many species withina matrix of material comprising many species of polymer. A- representsthe anionic or acidic groups that can include sulfonate, carboxylate, orother similar functional group. M+ represents the counter ion and mayinclude H+, Li+, Na+, or similar cation. Letters (p-z) accompanied byparenthesis or brackets represent repeat units that can range from 0 toany integer value. Any polymer containing any combination of Carbon (C),Fluorine (F), Sulfur (S), Oxygen (O), Hydrogen (H), Nitrogen (N),Phosphorous (P), or any similar element, which may be used to create anionic exchange membrane may also be utilized.

Ion exchange membranes can be constructed from polymers includingperfluorinated ionomers (1&2), polyphosphazene based ionomers (3),polystyrene based ionomers (4), polystyrene based block-co-polymerionomers (5), and poly(arlyene ether sulfone) based ionomers (6).

Total acid content for ionic exchange membranes may range from about0.57 to about 3.5 meq/g. In some embodiments, the total acid content forionic exchange is from about 0.57 to about 4.0 meq/g. In someembodiments, the total acid content for ionic exchange is from about0.57 to about 3.0 meq/g. In some embodiments, the total acid content forionic exchange is from about 0.57 to about 2.9 meq/g. In someembodiments, the total acid content for ionic exchange is from about0.57 to about 2.8 meq/g. In some embodiments, the total acid content forionic exchange is from about 0.57 to about 2.7 meq/g. In someembodiments, the total acid content for ionic exchange is from about0.57 to about 2.6 meq/g. In some embodiments, the total acid content forionic exchange is from about 0.57 to about 2.5 meq/g. In someembodiments, the total acid content for ionic exchange is from about0.57 to about 2.4 meq/g. In some embodiments, the total acid content forionic exchange is from about 0.57 to about 2.3 meq/g. In someembodiments, the total acid content for ionic exchange is from about0.57 to about 2.2 meq/g. In some embodiments, the total acid content forionic exchange is from about 0.57 to about 2.1 meq/g. In someembodiments, the total acid content for ionic exchange is from about0.57 to about 2.0 meq/g.

Membranes constructed from these ionomers may range in thickness fromabout 0.025 to about 0.69 mm in thickness. In some embodiments themembrane is from about 0.001 to about 069 mm in thickness. In someembodiments the membrane is from about 0.001 to about 068 mm inthickness. In some embodiments the membrane is from about 0.001 to about067 mm in thickness.

In some embodiments the membrane is from about 0.001 to about 066 mm inthickness. In some embodiments the membrane is from about 0.001 to about065 mm in thickness. In some embodiments the membrane is from about0.001 to about 064 mm in thickness. In some embodiments the membrane isfrom about 0.001 to about 063 mm in thickness. In some embodiments themembrane is from about 0.001 to about 062 mm in thickness. In someembodiments the membrane is from about 0.001 to about 061 mm inthickness. In some embodiments the membrane is from about 0.001 to about060 mm in thickness. In some embodiments the membrane is from about0.001 to about 059 mm in thickness. In some embodiments the membrane isfrom about 0.001 to about 058 mm in thickness. In some embodiments themembrane is from about 0.001 to about 050 mm in thickness. In someembodiments the membrane is from about 0.001 to about 040 mm inthickness. In some embodiments the membrane is from about 0.001 to about030 mm in thickness. In some embodiments the membrane is from about0.001 to about 020 mm in thickness. In some embodiments the membrane isfrom about 0.001 to about 010 mm in thickness. In some embodiments themembrane is from about 0.025 to about 065 mm in thickness. In someembodiments the membrane is from about 0.025 to about 064 mm inthickness. In some embodiments the membrane is from about 0.025 to about063 mm in thickness. In some embodiments the membrane is from about0.025 to about 062 mm in thickness. In some embodiments the membrane isfrom about 0.025 to about 061 mm in thickness. In some embodiments themembrane is from about 0.025 to about 060 mm in thickness. In someembodiments the membrane is from about 0.025 to about 059 mm inthickness. In some embodiments the membrane is from about 0.025 to about058 mm in thickness. In some embodiments the membrane is from about0.025 to about 050 mm in thickness. In some embodiments the membrane isfrom about 0.025 to about 040 mm in thickness. In some embodiments themembrane is from about 0.025 to about 030 mm in thickness. In someembodiments the membrane is from about 0.025 to about 020 mm inthickness. In some embodiments the membrane is from about 0.025 to about010 mm in thickness.

Higher total acid content and smaller membrane thickness leads to fasterdiffusion times. Membranes may be formed through extrusion casting, dropcasting, hot pressing, or similar method.

Catridges and Disposable Devices

The bio sensor, device, system, and or test strip may be or comprise acartridge. In some embodiments, the catridge is disposable after one useor can be used more than once per ammonia or ammonium ion detectionevent. In some embodiments, the catridge comprises a plurality ofmicrofluidic conduits in fluid communication with a storage portion, amixing portion and a readout portion of the catridge. The storageportion comprises a plurality of compartments that store one or acombination of indophenol reagents either crystalized, dried,lyophilized or in solution. In some embodiments, the compartments may bepartitioned from an adjacent conduit by plastic wall or other inertmaterial. The mixing portion of the catridge comprises a trunk-shapedconduit where one or more reagents being stored mix after they arereleased from the storage portion of the device. The reagents may mixwith a sample and/or each other at different points in the microfluidicchannels adajacent to the storage portion of the device. In someembodiments of the device the readout portion of the microfluidicconduits is adjacent to the mixing portion of the device. In someembodiments of the device, the cartridge comprises only a storageportion and a readout portion, wherein the readout portion comprises amicrofluidic conduit configured to align to an instrucment that measuresthe amount of ammonia or ammonium in a sample but also allows mixing ofsamples prior to any detection or quantification step takes placethrough the instrument. In some embodiments, the catridge does notcomprise an instrument for detection of the amount of ammonia orammonium ion in an sample (spectrophotometer), but is configured toalign the readout portion of the catridge to a instrument capable ofdetermining the amount of ammonia or ammonium ion in a sample. In someembodiments, the catridge comprises an instrument for detection of theamount of ammonia or ammonium ion in an sample, such as a photodiode. Insome embodiments, the catridge comprises readout portion comprisingmicrofluidic conduits for detection or quantification adjacent to themixing portion of the device. In some embodiments, the catridgecomprises an instrument for detection of the amount of ammonia orammonium ion in an sample, such as a photodiode, such instrumentcomprising a light source aligned to or with the readout portion of thedevice such that light from the light source may penetrate the readoutportion and such instrument may detct the presence, absence orabsorbance of wavrelength of light in the readout portion.

In some embodiments, the catridge comprises a microfluidic circuitcomprising a storage portion in fluid communication with a mixingportion which is also in fluid communication with a readout portion.Fluid in such an embodiment is designed to flow from the storage portionto the mixing portion, and from the mixing portion to the readoutportion of the catridge. In some embodiments the storage portioncomprises one compartment for each indophenol reagent. In someembodiments, the storage portion comprises a first compartmentcomprising a hypohalite (such as hypochlorite), a second compartmentcomprising an basic buffer (such as NaOH), and a third compartmentcomprising at least one indophenol reagent or indophenol relatedcompound (such as 2-phenylphenol). In some embodiments, the storageportion comprises a fourth compartment comprising a catalyst or couplingreagent (such as Sodium Nitroprusside). In some embodiments, the storageportion comprises a fifth compartment comprising an alkali buffer (suchas sodium acetate or calcium acetate or zinc acetate). In someembodments, the catridge comprises a fluid exchange opening between amicrofluidic conduit the compartment comprising a an alkali buffer (suchas sodium acetate or calcium acetate or zinc acetate). In someembodiments, a membrane disclosed herein is positioned over at least aportion of the fluid exchange opening such that when a sample comes incontact with the alkali buffer, ammonia can be transported across themembrane into the adjacent microfluidic conduit.

In some embodiments, the storage portion comprises a compartmentoptionally comprising an electrode. In some embodiments the compartmentoptionally comprising an electrode is adjacent to a compartmentcomprising the alkali buffer in solid or liquid phase, such compartmenthaving an opening through which a sample may be deposited into thecatridge from a point exterior to the cartridge. In some embodiments,the catridge comprises a sixth compartment comprising an opening andoptionally comprising an electrode, such compartment having an openingthrough which a sample may be deposited into the catridge from a pointexterior to the cartridge. In some embodiments, the catridge comprises asixth compartment comprising an opening and optionally comprising anelectrode, such compartment having an opening through which a sample maybe deposited into the catridge from a point exterior to the cartridge;wherein the catridge further comprises a a compartment comprising analkali buffer that is positioned at or substantially near thecompartment comprising the opening, such that, upon inserting a sampleinto the compartment with an opening, the alkali buffer is transportedto the compartmenr comprising the opening and mixes with the sample.

In some embodiments, a compartment has a volume of no more than about100 microliters of fluid. In some embodiments, one or more compartmentsin the catridge has a volume of no more than about 100 microliters offluid. In some embodiments, one or more compartments in the catridge hasa volume of no more than about 90 microliters of fluid. In someembodiments, one or more compartments in the catridge has a volume of nomore than about 80 microliters of fluid. In some embodiments, one ormore compartments in the catridge has a volume of no more than about 70microliters of fluid. In some embodiments, one or more compartments inthe catridge has a volume of no more than about 60 microliters of fluid.In some embodiments, one or more compartments in the catridge has avolume of no more than about 50 microliters of fluid. In someembodiments, one or more compartments in the catridge has a volume of nomore than about 40 microliters of fluid. In some embodiments, one ormore compartments in the catridge has a volume of no more than about 30microliters of fluid. In some embodiments, one or more compartments inthe catridge has a volume of no more than about 20 microliters of fluid.In some embodiments, one or more compartments in the catridge has avolume of no more than about 10 microliters of fluid.

FIGS. 24 through 28 depict an embodiment of the invention that is acatridge. one half of the ctaridge is depicted in FIG. 24 while theopposite facing half of the cartridge is depicted in FIG>25. The twohalves of the catridge may be secured together by one or a plurality ofmicrscrews, dowels or fastners. The two halves of the cartridge may bemade out of one or a plurality of inert materials such as a plasticand/or glass.

The catridge half disclosed in FIG. 24 comprises a first, second, third,fourth and fifth storage compartment. FIG. 24 depicts a first, second,third, fourth and fifth compartment (labeled 1, 2, 3, 4, and 5respectively) that define a volume immediately adjacent to, butpartitioned from, a microfluic conduit on a bottom half of thecartridge. The partition is delineated by the small solid dash bisectingthe space between the compartment and the microfluidic conduit (labeled10, 11, 12, 13, and 14 for each of the compartments 1, 2, 3, 4, and 5respectively. In this embodiment, the first compartment compriseshypohalite, the second compartment comprises an basic buffer, the thirdcompartment comprises a catalyst, the fourth compartment comprises aindophenol reagent (such as a phenolic compound), and the fifthcompartment comprises an alkali buffer, which, if in aqueous solution,may be at a concentration from about 500 mM to about 1 M sodium acetate.The storage portion of the microfluidic circuit comprises the storagepoints 1, 2, 3, 4, 5, and 6. Any membrnace disclosed herein may beplaced at or near position 14 such that, upon introduction of a samplesuch as whole blood in the compartment 6 of FIG. 25, mixing of thereagents can occur. Fluid from compartment 5 is mixed with the sampleand ammonia ions in solution may be transferred from 5 and 6 into themixing portion of the device 20 across the membrance. The reagents incompartments 1 through 4 are also released such that after a period ofabout 4-5 seconds, all reagents have entered the mixing portion of thedevice 20. The upper branched portion of the mixing portion 20 mixes theindophenol reagents contained in the compartments 1, 2, 3 and 4 whilethe ammonia from the sample and to buffer in 5 and 6 mix in the lowertrunk of the cartridge. Once in use, FIG. 26 depicts the anticipatedflow of fluid from each compartment to the mixing portion of thecartridge. Lighter shades of grey show the bolus of reagents from eachcompartment as they travel at 0 seconds (i); at 13 second (ii); and 24seconds (iii) through the microfluid circuit. After mixing is completein the mixing portion of the device, all reagents mix in the portion ofthe mixing portion closest proximae to the readout portion 25 of thecircuit. At the readout portion of the device, the catridge may have anopening though which light may travel and expose the fluid to a certainmeasurable wavelength of light. An instrument such as a photodiode maybe present near or adjacent to the readout portion of the device so thatmeasurements of absorbance may be taken.

In some embodiments, the catridge comprises at least one electrode thatdetects the presence or absence of ammonia or ammonium ion in a samplein a vessel configured to receive a sample from a point external to thecatridge. Once the electrode is activated by the presence of a sample,the storage portion of the cartridge open and release their contentssuch that a solution from each compartment is released into the mixingportion of the microfluidic conduits. The microfluidic conduits are of alength sufficient to mix all of the reagents from each compartment suchthat, by the time total fluid volume of reactants reach the readoutportion of the catridge, an indophenol reaction has taken place and anindophenol reaction product (such as indophenol or an indophenol relatedcompound) have formed in the microfluidic conduits.

TABLE 3 Examples of Indophenol Reagent Concentration Ranges ReagentRange 2-phenylphenol From about 50 to about 70 mmol/liter SodiumNitroprusside About 7 micromoles/liter Sodium Hydroxide From about 50 toabout 500 mmol/liter Sodium Hypochlorite From about 50 to about 120mmol/liter Sodium/Calcium Acetate From about 0.5 to about 1 mol/liter

Hydrogel

The bio sensor comprises a hydrogel in some embodiments. The hydrogelmay be a cross-linked polymeric material that swells in water but doesnot dissolve. It is envisioned that the hydrogel may be capable ofabsorbing at least about 1 to about 10 times, and in one embodiment atleast about 100 times, its own weight of a liquid. The hydrogel chosenfor use in the biosensor should depend directly on the method offunctionalization. It is envisioned that the hydrogel may bebiocompatible. In some embodiments, the hydrogel comprises sodiumalginate. In some embodiments, the hydrogel comprises from about 0.1% toabout 5% alginate weight/volume. In some embodiments, the hydrogelcomprises from about 0.1% to about 4% alginate weight/volume. In someembodiments, the hydrogel comprises from about 0.1% to about 3% alginateweight/volume. In some embodiments, the hydrogel comprises from about0.1% to about 2% alginate weight/volume. In some embodiments, thehydrogel comprises from about 0.1% to about 1% alginate weight/volume.In some embodiments, the hydrogel comprises from about 0.1% to about 1%alginate weight/volume. In some embodiments, the hydrogel comprises fromabout 0.2% to about 1% alginate weight/volume. In some embodiments, thehydrogel comprises sodium alginate. In some embodiments, the hydrogelcomprises from about 0.3% to about 1% alginate weight/volume. In someembodiments, the hydrogel comprises from about 0.4% to about 1% alginateweight/volume In some embodiments, the hydrogel comprises from about0.5% to about 1% alginate weight/volume. In some embodiments, thehydrogel comprises from about 0.6% to about 1% alginate weight/volume.In some embodiments, the hydrogel comprises from about 0.7% to about 1%alginate weight/volume. In some embodiments, the hydrogel comprises fromabout 0.8% to about 1% alginate weight/volume. In some embodiments, thehydrogel comprises from about 0.9% to about 1% alginate weight/volume.In some embodiments, the hydrogel comprises from about 1.0% to about3.0% alginate weight/volume. In some embodiments, the hydrogel comprisesfrom about 1.0% to about 2.0% alginate weight/volume. In someembodiments, the hydrogel comprises from about 1.0% to about 1.5%alginate weight/volume. In some embodiments, the hydrogel comprisesabout 1%, about 2%, or about 3% alginate weight/volume. In someembodiments, the hydrogel comprises sodium alginate. The aliginate maybe any individual polymer of alginate used in bulk form or repitivepattern of monomers, G blocks, M blocks, and/or GM blocks. In someembodiments the alignate comprises the formula:

where m and n are any positive integer. In some embodiments m and n areindepedently variable and any positive integer from about 1 to about1000. In some embodiments, the hydrogel may be polymerized from acrylicmonomers. The acrylic monomer may be one or a combination of thefollowing: acrylamido-glycolic acid, acrylamido-methyl-propa-ne-sulfonicacid, acrylamido-ethylphosphate, diethyl-aminoethyl-acrylamide-,trimethyl-amino-propyl-methacrylamide, N-octylacrylamide,N-phenyl-acrylamide and tert-butyl-acrylamide. In embodiments in whichthe device contains a cross-linking agent, exemplary cross-linkingagents may be N,N′-methylene-bis-acrylamide,N,N′-methylene-bismethacrylamide, diallyltatardiamide and poly(ethyleneglycol)dimethacrylate. Examples of suitable hydrogels may also includesilicon wafers, borosilicate glass substrates, 2-hydroxyethylmethacrylate (HEMA), N-Isopropylacrylamide (NIPAAm), and polyethyleneglycol (PEG).

The hydrogel may include any number of molecules. For example, thehydrogel may include a polymerized monomer or hydrogel a cross linkingagent and optionally a chemical or UV-light activated inducer agent.Examples of such monomers or dimers include vinyl acetates, vinylpyrrolidones, vinyl ethers, olefins, styrenes, vinyl chlorides,ethylenes, acrylates, methacrylates, nitriles, acrylamides, maleates,epoxies, epoxides, lactones, ethylene oxides, ethylene glycols,ethyloxazolines, amino acids, saccharides, proteins, anhydrides, amides,carbonates, phenylene oxides, acetals, sulfones, phenylene sulfides,esters, fluoropolymers, imides, amide-imides, etherimides, ionomers,aryletherketones, amines, phenols, acids, benzenes, cinnamates, azoles,silanes, chlorides, and epoxides, N,N′-methylenebisacrylamide,methylenebismethacrylamide ethyleneglycol-dimethacrylate,N,N′-methylenebisacrylamide, polyethyleneglycoldiacrylate (PEGDA),polyethyleneglycoldimethacrylate (PEGDMA), polyethyleneglycoldiacrylate(PEGDA), polyethyleneglycoldimethacrylate (PEGDMA), poly(vinylidenfluoride) (PVdF) based polymer, a polyacrylonitrile (PAN) based polymer,a polymethylmethacrylate (PMMA) based polymer, a polyvinyl chloride(PVC) based polymer, and a mixture of the poly(vinyliden fluoride)(PVdF) based polymer, polyacrylonitrile (PAN) based polymer,polymethylmethacrylate (PMMA) based polymer, and polyvinyl chloride(PVC) based polymer, and mixtures of any two or more thereof. IN someembodiments, the hydrogel does not comprise 3,4-dihydroxybenzoic acid(3, 4-DHB) or an analog thereof.

Cross linking agents and optionally the chemical or UV-light activatedinducer agent may include N,N′-methylenebisacrylamide,methylenebismethacrylamide ethyleneglycol-dimethacrylate and agentN,N′-methylenebisacrylamide. Irgacure 2959 (Ciba);2,2-dimethoxy-2-phenylacetophenone, 2-methoxy-2-phenylacetone,benzyl-dimethyl-ketal, ammonium sulfate, benzophenone, ethyl benzoinether, isopropyl benzoin ether, .alpha.-methyl benzoin ether, benzoinphenyl ether, 2,2-diethoxy acetophenone, 1,1-dichloro acetophenone,2-hydroxy-2-methyl-1-phenylpropane 1-on, 1-hydroxy cyclohexyl phenylketone, antraquinone, 2-ethyl antraquinone, 2-chloroantraquinone,tioxantone, isopropyltioxantone, chloro tioxantone,2,2-chlorobenzophenone, benzyl benzoate, and benzoyl benzoate, TEMED,and ammonium persulfate (APS). In some embodiments, hydrogel comprises aprotein, peptide, glycoprotein, proteoglycans, glycosaminoglycans,and/or carbohydrate that is secreted by cells into the extracellularenvironment. In some embodiments, the secreted protein, peptide,glycoprotein, proteoglycans, glycosamainoglycans, and/or carbohydrate,or structures composed thereof.

In some embodiments, the disclosure relates to a coated biosensor devicecomprising at least one coating, wherein the biosensor comprises ametabolic enzyme covalently bound or immobilized to the coating, whereinthe metabolic enzyme shares at least 70% sequence identify to SEQ IDNO:1 or SEQ ID NO:2 or shares at least 70% sequence identify tofunctional fragments of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments,the disclosure relates to a coated biosensor device comprising at leastone coating, wherein the biosensor comprises a metabolic enzymecovalently bound or immobilized within the coating, wherein the coatingcomprises a composition comprising a hydrogel matrix, said matrixcomprising any one or combination of: alginate, trehalose, at least oneelectron mediator, and at lest one reduction agent. In some embodiments,the disclosure relates to a coated biosensor device comprising at leastone coating, wherein the biosensor comprises a metabolic enzymecovalently bound or immobilized to the coating, wherein the coatingcomprises a composition comprising a hydrogel matrix, said matrixcomprising any one or combination of: poly(ethylene glycol)dimethyacrylate with a molecular weight of about 1000 (PEGDMA-1000),2-hydroxy-2 methyl propiophenone (HMPP) and at least one acrylate,wherein the acrylate is selected from the group consisting ofmethacrylic acid (MAA) and methyl methacrylate (MMA), wherein the ratioof PEGDMA:Acrylate is from about 10:90 mol % to about 70:30 mol %, andsaid HMPP is at a concentration of from about 0.2% to about 0.6%, totalweight.

In some embodiments, the hydrogel solution prior to curing comprisestrehalose or an analog thereof at a concentration from about 1 nM toabout 999 mM. In some embodiments, the hydrogel solution prior to curingcomprises trehalose at a concentration from about 1 μM to about 10 mM.In some embodiments, the hydrogel solution prior to curing comprisestrehalose at a concentration from about 1 μM to about 9 mM. In someembodiments, the hydrogel solution prior to curing comprises trehaloseat a concentration from about 1 μM to about 8 mM. In some emboidiments,the hydrogel solution prior to curing comprises trehalose at aconcentration from about 1 μM to about 7 mM. In some embodiments, thehydrogel solution prior to curing comprises trehalose at a concentrationfrom about 1 μM to about 6 mM. In some embodiments, the hydrogelsolution prior to curing comprises trehalose at a concentration fromabout 1 μM to about 5 mM. In some emboidiments, the hydrogel solutionprior to curing comprises trehalose at a concentration from about 1 μMto about 4 mM. In some emboidiments, the hydrogel solution prior tocuring comprises trehalose at a concentration from about 1 μM to about 3mM. In some embodiments, the hydrogel solution prior to curing comprisestrehalose at a concentration from about 1 μM to about 2 mM. In someembodiments, the hydrogel solution prior to curing comprises trehaloseat a concentration from about 1 μM to about 1 mM. In some embodiments,the hydrogel solution prior to curing comprises trehalose at aconcentration from about 10 μM to about 1 mM. In some embodiments, thehydrogel solution prior to curing comprises trehalose at a concentrationfrom about 100 μM to about 1 mM. In some embodiments, the hydrogelsolution prior to curing comprises trehalose at a concentration fromabout 200 μM to about 1 mM. In some embodiments, the hydrogel solutionprior to curing comprises trehalose at a concentration from about 300 μMto about 1 mM. In some embodiments, the hydrogel solution prior tocuring comprises trehalose at a concentration from about 400 μM to about1 mM. In some embodiments, the hydrogel solution prior to curingcomprises trehalose at a concentration from about 500 μM to about 1 mM.In some embodiments, the hydrogel solution prior to curing comprisestrehalose at a concentration from about 600 μM to about 1 mM. In someembodiments, the hydrogel solution prior to curing comprises trehaloseat a concentration from about 700 μM to about 1 mM. In some embodiments,the hydrogel solution prior to curing comprises trehalose at aconcentration from about 800 μM to about 1 mM. In some embodiments, thehydrogel solution (prior to contacting with the electrode) comprisestrehalose at a concentration from about 900 μM to about 1 mM.

Enzymes

Any one or more metabolic enzymes may be chosen to used with the presentdisclosure. Metabolic enzymes that can be used individually or incombination with the biosensor, system or test strip disclosed hereininclude: any bacterial clone of phenylalanine dehydrogenase, histidineammonia lyase, mistidine oxidase. pheylalanine lyase, glutamatedehydrogenase. In some embodiments the enzyme is chosen from any one orcombination of enzymes disclosed below or their respective functionalfragments that are at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% homoglous to the full-length enzyme or nucleic acid encoding suchenzyme.

Organism Enzyme GenBank Accession No SEQ ID NO Thermoactinomycesphenylalanine D00631.1  2 intermedius dehydrogenase Solanum lycopersicumphenylalanine ammonia- XM_004246602  7 lyase Thermoactinomycesphenylalanine DD421709.1  8 intermedius dehydrogenaseCaenorhabditis remanei phenylalanine XM_003102740  9 dehydrogenaseArabidopsis thaliana glutamate dehydrogenase NM_121822.3 10Spirochaeta africana Hisitidine ammonia lyase NC_017098.1 SEQ ID NO: 2MRDVFEMMDRYGHEQVIFCRHPQTGLKAIIALHNTTAGPALGGCRMIPYASTDEALEDVLRLSKGMTYKCSLADVDFGGGKMVIIGDPKKDKSPELFRVIGRFVGGLNGRFYTGTDMGTNPEDFVHAARESKSFAGLPKSYGGKGDTSIPTALGVFHGMRATARFLWGTDQLKGRVVAIQGVGKVGERLLQLLVEVGAYCKIADIDSVRCEQLKEKYGDKVQLVDVNRIHKESCDIFSPCAKGGVVNDDTIDEFRCLAIVGSANNQLVEDRHGALLQKRSICYAPDYLVNAGGLIQVADELEGFHEERVLAKTEAIYDMVLDIFHRAKNENITTCEAADRIVMERLKKLTDIRRILLEDPRNSARRSEQ ID NO: 7MASSIVQNGHVNGEAMDLCKKSINVNDPLNWEMAAESLRGSHLDEVKKMVDEFRKPIVKLGGETLTVAQVASIANVDNKSNGVKVELSESARAGVKASSDWVMDSMGKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESSHTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKLINSNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPNGEKLNAEEAFRVAGVTSGFFELQPKEGLALVNGTAVGSGMASMVLFESNILAVMSEVLSAIFAEVMNGKPEFTDYLTHKLKHHPGQIEAAAIMEHILDGSSYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRAATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNTRLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGLKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRSAVKNTVSQVAKRTLTMGANGELHPARFCEKELLRVVDREYVFAYADDPCSSTYPLMQKLRQVLVDHAMKNGESEKNVNSSIFQKIVAFEDELKAVLPKEVESARAVVESGNPAIPNRITECRSYPLYRLVRQELGSELLTGEKVRSPGEEIDKVFTAMCNGQIIDPLLECLKSWNGAPLPIC SEQ ID NO: 8     atgcgcgacg tgtttgaaat gatggaccgc tatggccacg agcaggtcat tttttgccgt  61 catccgcaaa ccggtctcaa agcgatcatc gccttgcata atacaaccgc ggggccggct 121 ttgggtggat gccgcatgat cccgtatgct tcgacggacg aagccttgga ggatgttttg 181 cggttgtcca aaggcatgac ctataaatgc agtctggcgg atgtggactt tggcggggga 241 aaaatggtta tcatcggcga tccgaaaaaa gataaatcgc cggagttgtt tcgcgtgatc 301 ggccgttttg tgggcgggtt aaacggccgt ttctataccg gaaccgacat gggaaccaat 361 ccggaagatt ttgtccatgc cgccagggaa tcgaaatctt ttgccggatt gccgaaatcg 421 tacggcggaa agggggacac atccattccc accgcgctcg gggtgtttca cggaatgcgg 481 gccaccgccc ggtttttatg ggggacggat cagctgaaag ggcgtgtggt tgccatccaa 541 ggagtcggca aggtgggaga gcgcttgttg cagcttttgg tcgaagtggg ggcttactgc 601 aaaattgccg acatcgattc ggtgcgatgc gaacagctga aagaaaagta tggcgacaag 661 gtccaattgg tggatgtgaa ccggattcac aaggagagtt gcgatatttt ctcgccttgc 721 gccaaaggcg gcgtggtcaa tgatgacacc attgacgagt tccgttgcct ggccattgtc 781 ggatccgcca acaaccaact ggtggaagac cggcatgggg cactgcttca aaaacggagc 841 atttgttatg cacccgatta tctggtgaat gccggcgggc tgattcaagt ggctgatgaa 901 ctggaaggct tccatgaaga gagagtgctc gccaaaaccg aagcgattta tgacatggtc 961 ctggatattt ttcaccgggc gaaaaatgag aatattacca cttgtgaggc agcggaccgg1021 atcgtgatgg agcgtttgaa aaagttaacc gatattcgcc ggatcttgtt ggaggatccc1081 cgcaacagcg caaggaggta a SEQ ID NO: 9MDFKAKLLAEMAKKRKAVSGLEVKEGGAKFVRGADLESKRTQEYEAKQEELAIKKRKADDEILQESTSRAKIVPEVPEAEFDEKTPMPEIHARLRQRGQPILLFGESELSVRKRLHQLEIEQPELNEGWENEMQTAMKFIGKEMDKAVVEGTADSATRHDIALPQGYEEDNWKSIEHASTLLGVGDEMKRDCDIILSICRYILARWARDLNDRPLDVKKTAQGMHEAAHHKQTTMHLKSLMTSMEKYNVNNDIRHHLAKICRLLVIERNYLEANNAYMEMAIGNAPWPVGVTRSGIHQRPGSAKAYVSNIAHVLNDETQRKYIQAFKRLMTKLQEYFPTDPSKSVEFVKKSV SEQ ID NO: 10MNALAATNRNFKLAARLLGLDSKLEKSLLIPFREIKVECTIPKDDGTLASFVGFRVQHDNARGPMKGGIRYHPEVDPDEVNALAQLMTWKTAVAKIPYGGAKGGIGCDPSKLSISELERLTRVFTQKIHDLIGIHTDVPAPDMGTGPQTMAWILDEYSKFHGYSPAVVTGKPIDLGGSLGRDAATGRGVMFGTEALLNEHGKTISGQRFVIQGFGNVGSWAAKLISEKGGKIVAVSDITGAIKNKDGIDIPALLKHTKEHRGVKGFDGADPIDPNSILVEDCDILVPAALGGVINRENANEIKAKFIIEAANHPTDPDADEILSKKGVVILPDIYANSGGVTVSYFEWVQNIQGFMWEEEKVNDELKTYMTRSFKDLKEMCKTHSCDLRMGAFTLGVNRVAQATILRGWGAMNTVTNQWKAVDIFTQIRDHEQVVFCNDKNTGLKAIIAIHDTTLGPALGGCRMYPYATVEDALFDVLRLSKGMTYKCLAADVDFGGGKAVIIGDPHKDKTPELFRAFGQFVESLNGRFYTGTDMGTTPDDFVHAMKETNCIVGVPEEYGGSGDSSVPTALGVIYGIQATNKVIWGSDELHGKTYAIQGLGKVGRKVAERLLKEGADLYVCDIHPTAIEAIVSYAKKLGANVKVVQGTEIYRTDADIFVPCAFGNVVNDNTIHVLKVKAIVGSANNQLLDVRHGQLLKEKGILYAPDYIVNAGGLIQVADELYGLNKERVLQKTKAIYSTLLHIYSRAEADHITTIEAANRFCEERLQQRSRRNDFFTHRKQPKWDIRR(SEQ ID NO: 1).

Solid Support

There are many forms of ammonia- or ammonium ion-measuring devices; onecommon type is represented by hand-held electronic meters which receiveblood samples via enzyme-based test strips. In using these systems, thepatient may for example lances a finger or alternate body site to obtaina blood sample, the strip is inserted into a test strip opening in themeter housing, the sample is applied to the test strip and theelectronics in the meter convert a current generated by the enzymaticreaction in the test strip to a amino acid concentration value.

Solid supports of the disclosure may be solid state but are a flixblesubstrate. According to the disclosure, the interdigitated array or atleast one electrode is disposed proximal to, e.g., on, a flexiblesubstrate. To act as a flexible substrate, a material must be flexibleand also insulating, and is typically relatively thin. The substrateshould be capable of adhering components of an IDA, or additionalcomponents of a sensor, to its surface. Such thin, insulative, flexiblesubstrates are known in the art of flexible circuits and flex circuitphotolithography. “Flexible substrates” according to the presentdisclosure can be contrasted to non-flexible substrates used inintegrated circuit (IC) photolithography but not in flexible circuitphotolithography. Examples of non-flexible substrates used in ICphotolithography include silicon, aluminum oxide, and other ceramics.These non-flexible substrates are chosen to be processable to a veryflat surface. Typical flexible substrates for use in the disclosure areconstructed of thin plastic materials, e.g., polyester, especially hightemperature polyester materials; polyethylene naphthalate (PEN); andpolyimide, or mixtures of two or more of these. Polyimides are availablecommercially, for example under the trade name Kapton®, from I.E. duPontde Nemours and Company of Wilmington, Del. (duPont). Polyethylenenaphthalate is commercially available as Kaladex®, also from duPont. Aparticularly preferred flexible substrate is 7 mil thick Kaladex® film.

Interdigitated arrays of the disclosure can be used in applicationsgenerally known to incorporate electrodes, especially applications knownto involve interdigitated arrays of electrodes. Various applications areknown in the arts of electronics and electrochemistry, includingapplications relating to process and flow monitoring or control, andchemical analytical methods. The arrays may be particularly useful as acomponent of an electrochemical sensor, where there is added value,benefit, or cost efficiency, to the use of a flexible substrate, orwhere there is value, benefit, or cost efficiency in having aninterdigitated array of dimensions relatively larger than the dimensionsof interdigitated arrays conventionally disposed on non-flexiblesubstrates.

An interdigitated array of the disclosure can, for example, be includedin an electrochemical sensor (sometimes referred to as a “biosensor” orsimply “sensor”) used in electrochemical detection methods.Electrochemical detection methods operate on principles of electricityand chemistry, or electrochemistry, e.g., on principles of relating themagnitude of a current flowing through a substance, the resistance of asubstance, or a voltage across the substance given a known current, tothe presence of a chemical species within the substance. Some of thesemethods can be referred to as potentiometric, chronoamperometric, orimpedance, depending on how they are practiced, e.g., whether potentialdifference or electric current is controlled or measured. The methodsand sensors, including sensors of the disclosure, can measure currentflowing through a substance due directly or indirectly to the presenceof a particular chemical compound (e.g., an analyte or an electroactivecompound), such as a compound within blood, serum, interstitial fluid,or another bodily fluid, e.g., to identify levels of amino acids, bloodurea, nitrogen, cholesterol, lactate, and the like. Adaptations of someelectrochemical methods and electrochemical sensors, and features oftheir construction, electronics, and electrochemical operations, aredescribed, for example, in U.S. Pat. Nos. 5,698,083, 5,670,031,5,128,015, and 4,999,582, each of which is incorporated herein byreference.

In some embodiments, any of the above biosensor catridges, devices, ormethods comprise a volumne of anticoagulant. In some embodiments, thevolume of the anticoagulant disclosed herein in a volume of about 10microliters. In some embodiments, the volume of the anticoagulantdisclosed herein in a volume of about 20 microliters. In someembodiments, the volume of the anticoagulant disclosed herein in avolume of about 30 microliters. In some embodiments, the volume of theanticoagulant disclosed herein in a volume of about 40 microliters. Insome embodiments, the volume of the anticoagulant disclosed herein in avolume of about 50 microliters. In some embodiments, the volume of theanticoagulant disclosed herein in a volume of about 100 microliters.

In some embodiments, the methods disclosed herein comprise a step ofmixing a sample comprising blood with an anticoagulant such as heparin,Acenocoumarol, phenprocoumon, Atromentin, Brodifacoum, Phenindione,Coumadin or the like. In some embodiments the biosensor, catridge,device, or test strip comprise a mechanical shaker mechanism configuredto shake one or more volumes within the at least one vessel,microfluidic conduit, or mixing portion of the biosensor, catridge,device, or test strip.

Methods

The disclosure relates to a method of diagnosing or prognosing aclinical outcome of a subject with hyperammonemia or a hyperammoniarelated disorder, comprising contacting a sensor, system, or test stripdisclosed herein with a sample of bodily fluid, and quantifying a levelof ammonia or ammonium ion in the sample; and comparing the level ofamino acid in the sample to a threshold value of what is considerednormal level of amino acid level in the bodily fluid. In someembodiments, the method relates to to a method of diagnosing orprognosing a clinical outcome of a subject suspected of having or havingbeen previously diagnosed with hyerpammonemia or ahyperammonemia-related disorder and/or at least one aminoacidopathy.

In some embodiments, the method relates to to a method of diagnosing orprognosing a clinical outcome of a subject suspected of having or havingbeen previously diagnosed with at least one hyerpammonemia or ahyperammonemia-related disorder. The ranges of what ammonia or ammoniumion levels are considered normal for each age type are below in Table 4.If, after performing the quantification steps provided herein, theamount of ammonia or ammonium ion in the sample solution exceeds orfalls below the ranges provided, diet regimen, exercise regimen, and/ormedical treatment may be initiated or changed such that ammonia orammonium ion levels are monitored until the subject's levels havestabilized or fall within what is considered a healthy range.

TABLE 4 Ammonia Ranges Case Range Newborn - Healthy Less than 110micromoles/liter Newborn - Suspected Metabolic Greater than 200micromoles/liter Disorder Older than Newborn - Healthy 50-80micromoles/liter Older than Newborn - Suspected Greater than 100micromoles/liter Metabolic Disorder Hepatic Encephalopathy Greater than70 micromoles/liter

The disclosure relates to a method of detecting the presence or absenceor quantity of ammonia or ammonium related disorder in bodily fluids.The disclosure also relates to a method of quantifying the concentrationof ammonia or ammonium ion in bodily fluids of a subject. Quantificationcan occur at the point-of-care due to the quick enzymatic reactionreadout caused by the generation of a detectable current within acircuit after exposure of a sample from a subject to one or a pluralityof vessels comprising any one or combination of indophenol reagentsdisclosed herein. In some embodiments, the device or system describedherein may be utilized to detect if a person has abnormally high or lowlevels of ammonia in the blood, after which an electronic message ordisplay may then be provided to the user of the device or system oractivated on a display by one or more processors or microchips thatremotely or directly access one or more storage memories comprising oneor rmore concentration values of ammonia or ammonium ion in sample ofthe subject. In some embodiments, multiple concentration values may beobtained either simultaneously or in series, compared or analyzed by theone or more processors operably connected to the device or systemdisclosed herein. In some embodiments, multiple concentration values ofa subject over a time period may be compared or analyzed by the one ormore processors operably connected to the device or system disclosedherein, after which a message comprising the concentration value and/orthreshold values are displayed. In some embodiments, the messageoptionally includes a signal indicating that the subject should seekmedical treatment or alter diet to control ammonia or ammonium ionlevels in the subject.

The disclosure also relates to a method of diagnosing a subject with aliver dysfunction comprising: (a) contacting a sample of bodily fluidfrom a subject to the to the biosensor, system or test strip disclosedherein;

(b) quantifying one or more concentration values of ammonia in thesample;

(c) comparing the one or more concentration values of ammonia in thesample to a threshold value of ammonia concentration identified as beingin a healthy range; and

(d) identifying the subject as having a metabolic disease if the one ormore concentration values of ammonia in the sample exceed or fall belowthe threshold value. In some embodiments, if the sample is blood orwhole blood, the method comprises contacting the sample with ananticoagulant before or simultaneously with step (a).

The disclosure also relates to a method of diagnosing a subject withhyperammonemia comprising: (a) contacting a sample of bodily fluid froma subject to the to the biosensor, system or test strip disclosedherein;

(b) quantifying one or more concentration values of ammonia in thesample;

(c) comparing the one or more concentration values of ammonia in thesample to a threshold value of ammonia concentration identified as beingin a healthy range; and

(d) identifying the subject as having a metabolic disease if the one ormore concentration values of ammonia in the sample exceed or fall belowthe threshold value. In some embodiments, if the sample is blood orwhole blood, the method comprises contacting the sample with ananticoagulant before or simultaneously with step (a).

The disclosure also relates to a method of quantifying the amount ofamino acid in sample comprising: (a) contacting a sample of bodily fluidfrom a subject to the to the biosensor, system or test strip disclosedherein;

(b) quantifying one or more concentration values of ammonia in thesample;

(c) comparing the one or more concentration values of ammonia in thesample to a threshold value of ammonia concentration identifiedcorrelating to amino acid quantity; and

(d) identifying the amino acid levels if the one or more concentrationvalues of ammonia in the sample exceed or fall below the thresholdvalue. Any amnio acid may be detected using the reference informationfrom FIG. 14 wherein the biosensor, system or test strip disclosedherein comprises an enzyme disclosed herein or a functional fragmentthat has 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%sequence identity to any enzyme disclosed herein. One of ordinary skillin the art would know, for instance, that to detect the presence,absence, or quantity of amino acids listed on Table 5, one or morerecombinant or synthetic enzymes disclosed herein or a functionalfragment thereof that has 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% sequence identity to any sequence (either nucleic acid orencoded amino acid) disclosed herein.

In some embodiments, the phenolic reagent or indophenol reagent is usedin a range from about 50 to about 70 mmol/liter. In some embodiments,the phenolic reagent or indophenol reagent is used in a range from about52 to about 70 mmol/liter. In some embodiments, the phenolic reagent orindophenol reagent is used in a range from about 54 to about 70mmol/liter. In some embodiments, the phenolic reagent or indophenolreagent is used in a range from about 56 to about 70 mmol/liter. In someembodiments, the phenolic reagent or indophenol reagent is used in arange from about 58 to about 70 mmol/liter. In some embodiments, thephenolic reagent or indophenol reagent is used in a range from about 60to about 70 mmol/liter. In some embodiments, the phenolic reagent orindophenol reagent is used in a range from about 62 to about 70mmol/liter. In some embodiments, the phenolic reagent or indophenolreagent is used in a range from about 64 to about 70 mmol/liter. In someembodiments, the phenolic reagent or indophenol reagent is used in arange from about 66 to about 70 mmol/liter. In some embodiments, thephenolic reagent or indophenol reagent is used in a range from about 68to about 70 mmol/liter. In some embodiments, the phenolic reagent orindophenol reagent is used in a range from about 50 to about 68mmol/liter. In some embodiments, the phenolic reagent or indophenolreagent is used in a range from about 50 to about 66 mmol/liter. In someembodiments, the phenolic reagent or indophenol reagent is used in arange from about 50 to about 64 mmol/liter. In some embodiments, thephenolic reagent or indophenol reagent is used in a range from about 50to about 62 mmol/liter. In some embodiments, the phenolic reagent orindophenol reagent is used in a range from about 50 to about 60mmol/liter. In some embodiments, the phenolic reagent or indophenolreagent is used in a range from about 50 to about 58 mmol/liter. In someembodiments, the phenolic reagent or indophenol reagent is used in arange from about 50 to about 56 mmol/liter. In some embodiments, thephenolic reagent or indophenol reagent is used in a range from about 50to about 54 mmol/liter. In some embodiments, the phenolic reagent orindophenol reagent is used in a range from about 50 to about 52mmol/liter. In some embodiments, the phenolic reagent or indophenolreagent is used in concentration about 59 mmol/liter. In someembodiments, the phenolic reagent or indophenol reagent is2-phenylphenol.

In some embodiments, the catalyst is used in a concentration of about 7micromoles/liter. In some embodiments, the catalyst is sodiumnitroprusside.

In some embodiments, the basic buffer is used in a range from about 50to about 500 mmol/liter. In some embodiments, the basic buffer is usedin a range from about 120 to about 500 mmol/liter. In some embodiments,the basic buffer is used in a range from about 140 to about 500mmol/liter. In some embodiments, the basic buffer is used in a rangefrom about 160 to about 500 mmol/liter. In some embodiments, the basicbuffer is used in a range from about 180 to about 500 mmol/liter. Insome embodiments, the basic buffer is used in a range from about 200 toabout 500 mmol/liter. In some embodiments, the basic buffer is used in arange from about 220 to about 500 mmol/liter. In some embodiments, thebasic buffer is used in a range from about 240 to about 500 mmol/liter.In some embodiments, the basic buffer is used in a range from about 260to about 500 mmol/liter. In some embodiments, the basic buffer is usedin a range from about 280 to about 500 mmol/liter. In some embodiments,the basic buffer is used in a range from about 300 to about 500mmol/liter. In some embodiments, the basic buffer is used in a rangefrom about 320 to about 500 mmol/liter. In some embodiments, the basicbuffer is used in a range from about 340 to about 500 mmol/liter. Insome embodiments, the basic buffer is used in a range from about 360 toabout 500 mmol/liter. In some embodiments, the basic buffer is used in arange from about 380 to about 500 mmol/liter. In some embodiments, thebasic buffer is used in a range from about 400 to about 500 mmol/liter.In some embodiments, the basic buffer is used in a range from about 420to about 500 mmol/liter. In some embodiments, the basic buffer is usedin a range from about 440 to about 500 mmol/liter. In some embodiments,the basic buffer is used in a range from about 460 to about 500mmol/liter. In some embodiments, the basic buffer is used in a rangefrom about 480 to about 500 mmol/liter. In some embodiments, the basicbuffer is used in a range from about 100 to about 480 mmol/liter. Insome embodiments, the basic buffer is used in a range from about 100 toabout 460 mmol/liter. In some embodiments, the basic buffer is used in arange from about 100 to about 440 mmol/liter. In some embodiments, thebasic buffer is used in a range from about 100 to about 420 mmol/liter.In some embodiments, the basic buffer is used in a range from about 100to about 400 mmol/liter. In some embodiments, the basic buffer is usedin a range from about 100 to about 380 mmol/liter. In some embodiments,the basic buffer is used in a range from about 100 to about 360mmol/liter. In some embodiments, the basic buffer is used in a rangefrom about 100 to about 340 mmol/liter. In some embodiments, the basicbuffer is used in a range from about 100 to about 320 mmol/liter. Insome embodiments, the basic buffer is used in a range from about 100 toabout 300 mmol/liter. In some embodiments, the basic buffer is used in arange from about 100 to about 280 mmol/liter. In some embodiments, thebasic buffer is used in a range from about 100 to about 260 mmol/liter.In some embodiments, the basic buffer is used in a range from about 100to about 240 mmol/liter. In some embodiments, the basic buffer is usedin a range from about 100 to about 220 mmol/liter. In some embodiments,the basic buffer is used in a range from about 100 to about 200mmol/liter. In some embodiments, the basic buffer is used in a rangefrom about 100 to about 180 mmol/liter. In some embodiments, the basicbuffer is used in a range from about 100 to about 160 mmol/liter. Insome embodiments, the basic buffer is used in a range from about 100 toabout 140 mmol/liter. In some embodiments, the basic buffer is used in arange from about 100 to about 120 mmol/liter. In some embodiments, thebasic buffer is used in a concentration about 50 mmol/liter. In someembodiments, the basic buffer is sodium hydroxide. In some embodiments,the basic buffer is used in a concentration about 100 mmol/liter. Insome embodiments, the basic buffer is sodium hydroxide. In someembodiments, the basic buffer is used in a concentration about 200mmol/liter. In some embodiments, the basic buffer is sodium hydroxide.In some embodiments, the basic buffer is used in a concentration about300 mmol/liter. In some embodiments, the basic buffer is sodiumhydroxide. In some embodiments, the basic buffer is used in aconcentration about 400 mmol/liter. In some embodiments, the basicbuffer is sodium hydroxide. In some embodiments, the basic buffer isused in a concentration about 500 mmol/liter. In some embodiments, thebasic buffer is sodium hydroxide.

In some embodiments, the hypohalite is used in a range from about 50 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 52 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 54 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 56 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 58 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 58 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 60 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 62 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 64 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 66 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 68 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 70 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 72 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 74 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 76 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 78 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 80 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 82 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 82 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 84 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 86 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 90 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 92 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 94 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 96 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 98 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 100 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 102 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 104 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 106 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 108 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 110 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 112 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 114 toabout 120 mmol/liter. In some embodiments, the hypohalite is used in arange from about 116 to about 120 mmol/liter. In some embodiments, thehypohalite is used in a range from about 118 to about 120 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 118 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 116 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 114 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 112 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 110 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 108 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 106 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 104 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 102 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 100 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 98 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 96 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 94 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 92 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 90 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 88 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 86 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 84 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 82 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 80 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 78 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 76 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 74 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 72 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 70 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 68 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 66 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 64 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 62 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 60 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 58 mmol/liter. In some embodiments, the hypohalite is used in arange from about 50 to about 56 mmol/liter. In some embodiments, thehypohalite is used in a range from about 50 to about 54 mmol/liter. Insome embodiments, the hypohalite is used in a range from about 50 toabout 52 mmol/liter. In some embodiments, the hypohalite is used in aconcentration about 100 mmol/liter. In some embodiments, the hypohaliteis sodium hypochlorite.

In some embodiments, the alkali buffer is used in a range from about 0.5to about 1.0 mol/liter. In some embodiments, the alkali buffer is usedin a range from about 0.6 to about 1.0 mol/liter. In some embodiments,the alkali buffer is used in a range from about 0.7 to about 1.0mol/liter. In some embodiments, the alkali buffer is used in a rangefrom about 0.8 to about 1.0 mol/liter. In some embodiments, the alkalibuffer is used in a range from about 0.9 to about 1.0 mol/liter. In someembodiments, the alkali buffer is used in a range from about 0.5 toabout 0.9 mol/liter. In some embodiments, the alkali buffer is used in arange from about 0.5 to about 0.8 mol/liter. In some embodiments, thealkali buffer is used in a range from about 0.5 to about 0.7 mol/liter.In some embodiments, the alkali buffer is used in a range from about 0.5to about 0.6 mol/liter. In some embodiments, the alkali buffer is usedin a concentration about 1.0 mol/liter. In some embodiments, the alkalibuffer is one or a combination of: calcium acetate, calcium chloride,zinc acetate, zinc chloride, or any equivalent mono, di, or tri, -valentsalt thereof. In some embodiments, the alkali buffer is sodium/calciumacetate. In some embodiments, the alkali buffer is used in a range fromabout 0.5 to about 0.6 mol/liter. In some embodiments, the alkali bufferis used in a concentration about 1.0 mol/liter. In some embodiments, thealkali buffer is one or a combination of: calcium acetate, calciumchloride, zinc acetate, zinc chloride, or any equivalent mono, di, ortri, -valent salt thereof. In some embodiments, the alkali buffer issodium/calcium acetate. In some embodiments, the alkali buffer is usedin a range from about 0.5 to about 0.6 mol/liter. In some embodiments,the alkali buffer is used in a concentration about 1.0 mol/liter. Insome embodiments, the alkali buffer is one or a combination of: calciumacetate, calcium chloride, zinc acetate, zinc chloride, or anyequivalent mono, di, or tri, -valent salt thereof. In some embodiments,the alkali buffer is sodium/calcium acetate. In some embodiments, thealkali buffer is used in a range from about 0.5 to about 0.6 mol/liter.In some embodiments, the alkali buffer is used in a concentration about1.0 mol/liter. In some embodiments, the alkali buffer is one or acombination of: calcium acetate, calcium chloride, zinc acetate, zincchloride, or any equivalent mono, di, or tri, -valent salt thereof. Insome embodiments, the alkali buffer is sodium/calcium acetate. In someembodiments, the alkali buffer is used in a range from about 0.5 toabout 0.6 mol/liter. In some embodiments, the alkali buffer is used in aconcentration about 1.0 mol/liter. In some embodiments, the alkalibuffer is one or a combination of: calcium acetate, calcium chloride,zinc acetate, zinc chloride, or any equivalent mono, di, or tri, -valentsalt thereof. In some embodiments, the alkali buffer is sodium/calciumacetate. In some embodiments, the alkali buffer is used in a range fromabout 0.5 to about 0.6 mol/liter. In some embodiments, the alkali bufferis used in a concentration about 0.5 mol/liter. In some embodiments, thealkali buffer is one or a combination of: calcium acetate, calciumchloride, zinc acetate, zinc chloride, or any equivalent mono, di, ortri, -valent salt thereof. In some embodiments, the alkali buffer issodium/calcium acetate. In some embodiments, the alkali buffer is usedin a concentration about 0.6 mol/liter. In some embodiments, the alkalibuffer is used in a concentration about 0.7 mol/liter. In someembodiments, the alkali buffer is used in a concentration about 0.8mol/liter. In some embodiments, the alkali buffer is used in aconcentration about 0.9 mol/liter. In some embodiments, the alkalibuffer is used in a concentration about 0.75 mol/liter.

The disclosure relates to a method of diagnosing liver dysfunction orhyperammonemia in a subject comprising:

-   -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) detecting the presence, absence, or quantity of ammonia;    -   (c) correlating the quantity of ammonia to the levels of amino        acid in the sample;    -   (d) diagnosing the subject as having liver dysfunction or        hyperammonemia if the ammonia levels are quantified as above        about 100 micromoles/liter of sample.

The disclosure relates to a method of diagnosing liver dysfunction orhyperammonemia in a subject comprising:

-   -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) detecting the presence, absence, or quantity of ammonia;    -   (c) correlating the quantity of ammonia to the levels of amino        acid in the sample;    -   (d) diagnosing the subject as having liver dysfunction or        hyperammonemia if the ammonia levels are quantified as above        about 90 micromoles/liter of sample.

The disclosure relates to a method of diagnosing liver dysfunction orhyperammonemia in a subject comprising:

-   -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) detecting the presence, absence, or quantity of ammonia;    -   (c) correlating the quantity of ammonia to the levels of amino        acid in the sample;    -   (d) diagnosing the subject as having liver dysfunction or        hyperammonemia if the ammonia levels are quantified as above        about 80 micromoles/liter of sample.

The disclosure relates to a method of diagnosing liver dysfunction orhyperammonemia in a subject comprising:

-   -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) detecting the presence, absence, or quantity of ammonia;    -   (c) correlating the quantity of ammonia to the levels of amino        acid in the sample;    -   (d) diagnosing the subject as having liver dysfunction or        hyperammonemia if the ammonia levels are quantified as above        about 70 micromoles/liter of sample.

A method of treating a subject with liver dysfunction or hyperammonemiacomprising:

-   -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) diagnosing the subject as having liver dysfunction or        hyperammonemia if the ammonia levels are quantified as above        about 70 micromoles/liter of sample; and    -   (c) treating the subject by administering steroids, arginine        supplements, sodium benzoate, phenylacetate, and/or a glucose        solution.        A method of treating a subject with liver dysfunction or        hyperammonemia comprising:    -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) diagnosing the subject as having liver dysfunction or        hyperammonemia if the ammonia levels are quantified as above        about 80 micromoles/liter of sample; and    -   (c) treating the subject by administering steroids, arginine        supplements, sodium benzoate, phenylacetate, and/or a glucose        solution.        A method of treating a subject with liver dysfunction or        hyperammonemia comprising:    -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) diagnosing the subject as having liver dysfunction or        hyperammonemia if the ammonia levels are quantified as above        about 90 micromoles/liter of sample; and    -   (c) treating the subject by administering steroids, arginine        supplements, sodium benzoate, phenylacetate, and/or a glucose        solution.        A method of treating a subject with liver dysfunction or        hyperammonemia comprising:    -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) diagnosing the subject as having liver dysfunction or        hyperammonemia if the ammonia levels are quantified as above        about 100 micromoles/liter of sample; and    -   (c) treating the subject by administering steroids, arginine        supplements, sodium benzoate, phenylacetate, and/or a glucose        solution.

In any of the above methods, the method comprises detecting the ammoniaor ammonium ion levels in whole blood, water, or a sample taken from amicroenvironment such as a test solution reconstituted from a swab takenfrom a microenvironment.

The disclosure relates to a method of diagnosing a metabolic disorder ina subject comprising:

-   -   (a) contacting a sample of the subject to a system, catridge,        test strip, biosensor or device disclosed herein;    -   (b) detecting the presence, absence, or quantity of ammonia;    -   (c) correlating the quantity of ammonia to the levels of amino        acid in the sample;    -   (d) diagnosing the subject as having a metabolic disorder if the        amino acid levels are quantified as above those levels set forth        in Table 1.

In some embodiments, any methods disclosed herein comprises takingmultiple steps of detecting the presence, absence, or quantity ofammonia in a sample by performing 1, 2, 3, or more tests simultaneouslyor in series.

In some embodiments, the step of detecting the presence, absence, orquantity of ammonia comprises detecting the wavelength emitted orabsorbed by a indophenol reaction product. In any of the above methods,the step of detecting the presence, absence, or quantity of ammoniacomprises detecting the wavelength emitted or absorbed by a indophonelreaction product by looking at the visible light in one or more vessels.In some embodiments, the step of detecting the presence, absence, orquantity of ammonia comprises detecting the wavelength absorbed by aindophenol reaction product wherein the wavelength from about 500 nm toabout 700 nm.

In some embodiments, any of the above methods, the step of detecting thepresence, absence, or quantity of ammonia comprises detecting thewavelength emitted or absorbed by a indophonel reaction product.

In some embodiments, any of the above methods do not comprise a step ofconverting liquid to a gas or any step involving gas chromatography.

In some embodiments, any of the above biosensor catridges, devices, ormethods comprise mixing a volume of any of the reagents disclosed hereinin a volume of from about 10 microliters to about 150 microliters. Insome embodiments, any of the above biosensor catridges, devices, ormethods comprise comprise mixing a volume of any of the reagentsdisclosed herein in a volume of from about 10 microliters to about 100microliters. In some embodiments, any of the above biosensor catridges,devices, or methods comprise a volume of any of the reagents disclosedherein in a volume of from about 10 microliters to about 150microliters. In some embodiments, any of the above biosensor catridges,devices, or methods comprise a volume of any of the reagents disclosedherein in a volume of about 10 microliters. In some embodiments, any ofthe above bio sensor catridges, devices, or methods comprise a volume ofany of the reagents disclosed herein in a volume of about 20microliters. In some embodiments, any of the above biosensor catridges,devices, or methods comprise a volume of any of the reagents disclosedherein in a volume of about 30 microliters. In some embodiments, any ofthe above biosensor catridges, devices, or methods comprise a volume ofany of the reagents disclosed herein in a volume of about 40microliters. In some embodiments, any of the above biosensor catridges,devices, or methods comprise a volume of any of the reagents disclosedherein in a volume of about 50 microliters.

In some embodiments, the disclosure relates to a computer-implementedmethod of quantifying ammonia or ammonium ions and/or amino acidconcentration in a sample.

In some embodiments, the disclosure relates to a system comprising aprocessor that performs a computer-implemented method of quantifyingamino acid concentration in a sample of a subject. In some embodiments,the system comprises a processor optinally located at a remote locationand accessible by internet connection, operably connected to a computerstorage memory that stores subejct's concentration values over time. Insome embodiments, the subject or the subject's healthcare provider mayaccesses the internet to communicate with a server linked to thecomputer storage memory. Subject data reports may be generated andobtained by the subject after initiating a retrieve command through theprocessor. In some embodiments, the system comprises a computerprogram-product that performs a function convert current signalsgenerated by a biosensor disclosed herein to concentration of aparticular amino acid and/or ammonia in a sample. In some embodiments,the disclosure relates to a system including at least one processor anda computer readable memory, said computer readable memory having storedthereon program code for quantifying amino acid concentration in asample of bodily fluid comprising: means for storing data associatedwith a subject; means for, responsive to receiving a level of currentresponse from a biosensor or its computer storage memory, presenting aconcentration value to a user as part of a user interface. In someembodiments, the user is the subject or healthcare provider of thesubject. In some embodiments, the disclosure relates to a system thatcomprises at least one processor, a program storage, such as memory, forstoring program code executable on the processor, and one or moreinput/output devices and/or interfaces, such as data communicationand/or peripheral devices and/or interfaces. In some embodiments, theuser device and computer system or systems are communicably connected bya data communication network, such as a Local Area Network (LAN), theInternet, or the like, which may also be connected to a number of otherclient and/or server computer systems. The user device and client and/orserver computer systems may further include appropriate operating systemsoftware.

The present disclosure relates generally to definition and/or use ofconcentration values that characterize a subject's modification ofbehavior. in some embodiments, the concentration values corresponding tothe concentration of amino acids in a sample of bodily fluid maycharacterize the degree to which a subject is advised to modify a dietor seek medical treatment.

In some embodiments, the present disclosure provides biosensors or teststrips for use in diagnostic assays. In some embodiments the biosensorand/or test strips are provided as part of a diagnostic or detectionkit. In certain embodiments, kits for use in accordance with the presentdisclosure may include one or more reference samples; instructions(e.g., for processing samples, for performing tests, for interpretingresults, etc.); media; and/or other reagents necessary for performingtests.

The disclosure provides a test strip comprising: a solid support, a atleast a first vessel in fluid communication with at least one conduit,wherein the test strip comprises a hydrogel disclosed herein. In someembodiments, the solid support is a slide optionally coated with apolymer. In some embodiments, the solid support is coated with apolymer. In some embodiments, the polymer is polyacrylamide. In someembodiments, the solid support is a material chosen from: polysterene(TCPS), glass, quarts, quartz glass, poly(ethylene terephthalate) (PET),polyethylene, polyvinyl difluoride (PVDF), polydimethylsiloxane (PDMS),polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA),polycarbonate, polyolefin, ethylene vinyl acetate, polypropylene,polysulfone, polytetrafluoroethylene, silicones, poly(meth)acrylic acid,polyamides, polyvinyl chloride, polyvinylphenol, and copolymers andmixtures thereof. In some embodiments, the test strip is a paperproduct. In some embodiments, the at least one electrode is attached tothe solid support.

According to some embodiments, the disclosure provides a softwarecomponent or other non-transitory computer program product that isencoded on a computer-readable storage medium, and which optionallyincludes instructions (such as a programmed script or the like) that,when executed, cause operations related to the calculation of amino acidconcentration values. In some embodiments, the computer program productis encoded on a computer-readable storage medium that, when executed:quantifies one or more ammonia or ammonium ion concentration values;normalizes the one or more ammonia or ammonium ion concentration valuesover a control set of data; creates an amino acid profile or signatureof a subject; and displays the profile or signature to a user of thecomputer program product. In some embodiments, the computer programproduct is encoded on a computer-readable storage medium that, whenexecuted: calculates one or more ammonia or ammonium ion concentrationvalues, normalizes the one or more ammonia or ammonium ion concentrationvalues, and creates an amino acid signature, wherein the computerprogram product optionally displays the amino acid signature and/or oneor more ammonia or ammonium ion concentration values on a displayoperated by a user. In some embodiments, the disclosure relates to anon-transitory computer program product encoded on a computer-readablestorage medium comprising instructions for: quantifying one or moreammonia or ammonium ion concentration values; and displaying the one ormore ammonia or ammonium ion concentration values to a user of thecomputer program product.

In some embodiments, the step of calculating one or more ammonia orammonium ion concentration values comprises quantifying an average andstandard deviation of counts on replicate trials of contacting thedevice or test strip with one or more samples of bodily fluids.

In some embodiments, the one or more hydrogel coated electrodes areattached to a solid phase support. In some embodiments, a solid phasesupport comprises any solid or semi-solid surface. In some embodiments,a solid phase comprises any traditional laboratory material for growingor maintaining cells in culture including petri dishes, beakers, flasks,test tubes, microtitre plates, and/or culture slides. In someembodiments, a solid phase comprises a glass slide, a plastic slide, apaper test strip, or combination thereof.

In some embodiments, the one or more hydrogel coated electrodes areattached to discrete addressable sites on a solid phase support. In someembodiments, a solid phase comprises polyamides, polyesters,polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g.polyvinylchloride), polycarbonate, polytetrafluoroethylene (PTFE),nitrocellulose, cotton, polyglycolic acid (PGA), cellulose, dextran,gelatin, glass, fluoropolymers, fluorinated ethylene propylene,polyvinylidene, polydimethylsiloxane, polystyrene, silicon substrates(such as fused silica, polysilicon, or single silicon crystals) orcombinations thereof.

In some embodiments, the disclosure relates to a catalogue of medicalrecords relating to a subject comprising test results from the one orplurality of methods described herein. Such catalogue, in someembodiments, being stoed on a computer readable medium being accessibleremotely through a wireless internet connection.

As described above, certain embodiments of the present disclosure may beused to distinguish between samples of bodily fluid obtained from asubject who does or is suspected of having an hyperammonemia and asubject who does not have a metabolic disease. This system ispotentially useful, for example, when testing whole blood samples of asubject to determine whether disease is present. Diagnosing a patientusing one or more ammonia or ammonium ion concentration values wouldinclude, for example, comparing one or more ammonia or ammonium ionconcentration values of a sample from a subject with the measuredreference values or threshold values of a subject.

Kits

In some embodiments, kits in accordance with the present disclosure maybe used to quantify amino acid concentration is samples of bodily fluid.

The disclosure further provides for a kit comprising one or a pluralityof containers that comprise one or a plurality of the polypeptides orfragments disclosed herein. In some embodiments, the kit comprises atest strip and/or a biosensor comprising a test strip, or anyanimal-based derivative of serum that enhances the culture orproliferation of cells. In some embodiments, the kit comprises: abiosensor disclosed herein, any test strip disclosed herein, and acomputer program product disclosed herein optionally comprisinginstructions to perform any one or more steps of any method disclosedherein. In some embodiments, the kit does not comprise cell media. Insome embodiments, the kit comprises a solid support comprising amembrane disclosed herein and/or embedded with at least one electrodedisclosed herein optionally comprising any one or combination of ahypohalite, an aqueous basic solution, and at least one compoundcomprising a phenyl group in one or a a pluarality of containers. Insome embodiments, the kit comprises a device to affix a hydrogel to asolid support.

The kit may contain two or more containers, packs, or dispenserstogether with instructions for preparation of an array. In someembodiments, the kit comprises at least one container comprising the biosensor or system described herein and a second container comprising asolution for maintenance, use, and/or storage of the biosensor such asstorage buffer. In some embodiments, the kit comprises a compositioncomprising any molecule disclosed herein in solution or lyophilized ordried and accompanied by a rehydration mixture. In some embodiments, themolecules and rehydration mixture may be in one or more additionalcontainers. In some embodiments, the kit comprises a compositioncomprising any one or combination of

The compositions included in the kit may be supplied in containers ofany sort such that the shelf-life of the different components arepreserved, and are not adsorbed or altered by the materials of thecontainer. For example, suitable containers include simple bottles thatmay be fabricated from glass, organic polymers, such as polycarbonate,polystyrene, polypropylene, polyethylene, ceramic, metal or any othermaterial typically employed to hold reagents or food; envelopes, thatmay consist of foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, and syringes. Thecontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components of thecompositions to mix. Removable membranes may be glass, plastic, rubber,or other inert material.

The kit may contain a bio sensor described herein and/or a test stripcomprising ahypohalite, an aqueous basic solution, and at least onecompound comprising a phenyl group. The kit may also contain a soldsupport such as a test strip comprising any membrane disclosed herein.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrates, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, zipdisc, videotape, audio tape, or other readable memory storage device.Detailed instructions may not be physically associated with the kit;instead, a user may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

The disclosure also provides a kit comprising: a biosesnsor comprising:a solid support and a plurality of electrodes, wherein at least oneelectrode comprises a hydrogel disclosed herein. in some embodiments,the hydrogel comprises an immobilized metabolic enzyme or a functionalfragment thereof; and optionally comprising at least one vesselcomprising a hyohalite, an aqueous basic buffer, in liquid or solidphase, and at least one compound comprising a phenyl group. In someembodiments, the kit further comprises at least one of the following: asample, and a set of instructions, optionally accessible remotelythrough an electronic medium.

Generally referring to FIGS. 1-7, a system, method, and apparatus forpoint of care hyperammonemia sensors may be described. In the exemplaryembodiments described by the figures, samples may be tested for ammonialevels, amino acid levels, or other compound levels by being in concertwith certain reagents to utilize an indophenol reaction. Color change inthe reaction may be measured and correspond to certain concentrations ofspecific compounds and molecules by manual comparison to an extensivecolor-matching sheet or automated electronic analysis with the use ofcalibration curves.

FIG. 1 shows one exemplary embodiment of a system demonstrating theability to detect ammonia levels in various samples. A well 100 may bemade of plastic, wood, metal, composite materials, or a combinationthereof. Additionally, well 100 may be comprised of synthetic compoundsor polymers, such as silicone. Well 100 may further be divided into twoor more sections, and may be separated by a membrane filter 105interposed in or near the center of welllOO. Membrane filter 105 may bemade of a cation exchange filter such as Nafion, shown in SupplementaryFig. A, or similar perfluorinated ionomers to allow for only the passageof small positively charged and neutral molecules between sections.Therefore, membrane filter 105 may be selected to allow for the passageof various molecules or biological components based on charge, size, orsimilar characteristics. Other membrane filters may consequently be usedfor desired functionality, such as acrylamide, poly(ethylene glycol)diacrylate, poly(2-hydroxylethyl methacrylate), poly(vinyl alcohol), orother similar polymeric hydrogels. The selection of membrane filter 105for a hyperammonemia sensor may depend on the membranes ability to allowfor the passage of molecules such as ammonia, and the ability to limitthe passage of proteins, amino acids, and other molecules or compounds.

Still referring to FIG. 1, reagent section 101 may contain reagents suchas phenol, 2-phenylphenol, sodium salicylate, other phenolic reagents orpolymers, or a combination thereof. Further, reagent section 101 mayalso contain bleach, hypochlorite, chloramine T, a similar anion, or acombination thereof, catalysts such as nitroprusside, and a basic buffersuch sodium hydroxide or potassium hydroxide to maintain alkaliconditions. Sample section 102 may contain serum, blood, plasma, orother liquid desired to be tested. Membrane filter 105 may only allowthe passage of ammonia from section 102 to section 101. A chemicalreaction, described in FIG. 3, may take place upon reception of ammoniaor similar molecule into section 101, turning the reagents a blue color,as shown in section 103. Section 104 may describe the tested sampleafter the reaction takes place. Color sheets may be available for aqualitative comparison between colors representing specific ammoniumconcentrations.

In order for the cation exchange membrane, such as Nafion, to be useablefor this application, a certain washing procedure and method may bedisclosed. The membrane may be washed in a hydrogen peroxide aqueoussolution, which may be at boiling temperatures. Additionally, themembrane may be washed in deionized water, ethylenediaminetetraaceticacid or other chelating agents, sulfuric acid, and other similar aqueousmaterials. The membrane may be exposed to extreme temperatures andpressures to further ensure washing.

FIG. 2 shows an exemplary embodiment of a device fitted with multiplewells. Wells 200 may be depressions or fossa in a mounting plate 203.Mounting plate 203 may be comprised of plastic, wood, metals, compositematerials, or a combination thereof. Additionally, mounting plate 203may be comprised of synthetic compounds or polymers, such as silicone.As shown, mounting plate 203 carries three wells 200, yet those skilledin the art may appreciate the ability for a mounting plate 203 to carrysubstantially more or fewer wells as desired. Membrane filter 205 may bemade of Nafion or similar membranes, and may be disposed of in anyangle, such as a vertical placement as shown in FIG. 2, a horizontalplacement, or a different angle as desired. Reagent section 201 may befilled with phenol, 2-phenylphenol, other phenolic reagents, or acombination thereof; bleach, hypochlorite, chloramine T, a similaranion, or a combination thereof; sodium hydroxide, potassium hydroxide,or a similar basic buffer to maintain alkali conditions; and one or morecatalysts, such as nitroprusside. Sample section 202 may be filled withserum, blood, plasma, or similar material desired to be tested. Thevarious wells 200 may be interconnected to facilitate the fluid flowbetween respective sections in order to test samples multiple times tofurther accuracy, or to test samples with multiple different membranefilters or reagents. Generally, if sample section 202 containssufficient levels of ammonia, the ammonia may diffuse through membranefilter 205 and into reagent section 202, which may allow the reaction tobe described in FIG. 3 to take place.

FIG. 3 shows exemplary reactions that may take place in a point of carehyperammonemia sensor, sometimes known as an indophenol reaction orBerthelot's Reaction. Reactions 300, 330, and 360 may take place upondiffusion of ammonia from one section of a well to another through amembrane filter, as described in FIGS. 1-2. Anion 302 may behypochlorite, as shown, bleach, calcium hypochlorite, sodiumhypochlorite, or other similar anions. Anion 302 may then react withammonia 301, and produce chloramine 303, or similar ammonia derivative.Chloramine 303 may then react with further reagents, such as phenol 331.A phenol-cholarmine intermediate 333 may further react with additionalphenol 331 molecules, producing indophenol 363 which may appear visiblyblue in color. Phenol 331 may also be replaced with 2-phenylphenol forfurther efficacy, with other phenolic reagents such as sodiumsalicylate, with phenol polymers, or with a combination thereof. Thecolor change in the reagent section of the well or depression maydemonstrate the presence of ammonia in the sample section.

FIG. 4 shows a further exemplary embodiment of a testing device,comprising of a microfluidic. The microfluidic 400 may be suitable forhome use in a similar fashion to blood glucose meters to provideongoing, rapid, reliable testing for hyperammonemia, variousaminoacidopathies, and other similar applications. The device 401 may bemanufactured of plastic, wood, metal, composites, or a combinationthereof, or a synthetic polymer or compound, such as silicone. A usermay use a lancet to excrete a small amount of blood from the tip of afinger or other location on the body, and apply a small amount of blood,serum, plasma, or similar component at opening of a conduit channel 402.The sample may be transported through conduit channel 402 by capillaryaction and reach sample section 403. Sample section 403 may be separatedfrom reagent section 404 by a cation exchange membrane 405, such asNafion, whereby allowing ammonia to diffuse through membrane 405 intoreagent section 404. Prior to the application of a blood sample, asqueezable reservoir 406 containing either dry or liquid bleach,hypochlorite, chloramine T, or similar anion may be manually orelectronically stimulated, allowing for the flow of bleach intointerposed reagent section 404. The bleach may be separate from reagentsin reagent section 404 to ensure accurate and timely chemical reactions.Reagent section 404 may contain liquid or dry components of reagentsdisclosed in FIGS. 1-3, such as phenol, 2-phenylphenol, other phenolicreagents, or a combination thereof; sodium hydroxide, potassiumhydroxide, or a similar basic buffer to maintain alkali conditions; andmay also contain one or more catalysts, such as nitroprusside. Upon thepresence of a certain level of ammonium in the sample, the reagentsection 404 may tum into a blue color, which may be compared to aseparate or included color schematic for the user to identify.

Still referring to FIG. 4, the microfluidic 400 may be used multipletimes or manufactured to be a single-use device. Additionally, changesmay be implemented to the design and range of chemicals used todetermine amino acid levels in samples. Those skilled in the art mayalso appreciate the ability for a device or similar device to conform tovarious biological or non-biological samples, such as saliva, urine,waste water, or perhaps various chemicals to be used in a laboratory ormedical setting.

In addition to the qualitative methods of determining presence or levelsof ammonia in applicable samples, a quantitative apparatus, system, andmethod may be disclosed.

FIG. 5 shows an exemplary flowchart of a sequence of events that maytake place to accurately and quantitatively identify the amount ofammonia in a sample, and is closely related to the exemplary apparatusand system disclosed in FIGS. 6-7. Additionally, those skilled in theart may appreciate that quantitative analysis in this, or a similarfashion, be added to any of the apparatuses or systems disclosed inFIGS. 1-4.

FIG. 5 therefore shows an exemplary flowchart of steps for aquantitative point of care hyperammonemia sensor. It may be appreciatedthat these steps may be interchangeable chronologically, may be alteredsignificantly, or eliminated while receiVmg similar results. Block 501may refer to a test strip of any size, similar to sizing of the testingstrips of blood glucose meters. The insertion mechanism block 501 teststrip may be manual or automated. Upon insertion into a device, block502 may further disclose the initiation of a series of events that maytake place under program control. A bleach reservoir may be opened,manually or automatically, into a reagent section within the device. Thereagent section, sample section, or both may contain reagents necessaryfor an indophenol reaction, or reagents used for diagnosingaminoacidopathies or similar diseases and conditions. Block 503 mayfurther disclose the application of a blood sample by way of lancetexcretion. The blood sample may be substituted for other biologicalsamples, which may then be transported through a conduit channel to asample section separated by a reagent section by a cation exchangemembrane, such as Nafion. Block 503 may further initiate a microchipunder program control which may serve as a timing device, allowing forconsistent timing between various steps. This microchip may direct aphotodiode or photoresistor to remain inactive for a desired duration toallow for an adequate period of time for certain reactions to takeplace.

Still referring to FIG. 5, Block 504 may further disclose the diffusionof ammonia or similar compound from sample section to reagent section toinitiate any reaction. After a determined period of time, the reagentsection may turn blue in the presence of ammonia. The degree ofcoloration may be dependent on the amount of ammonia in the samplesection, which will allow for accurate quantitative analysis. Block 505may further disclose the initiation of a photodiode or photoresistornear the reagent section to measure the degree of coloration. Thephotodiode or photoresistor may change the current of the system basedon the coloration, whereby block 506 may disclose the step of convertingphotodiode or photoresistor signal from an analog to a digital signal.Block 507 may further disclose the reception of a digital signal to amicrochip under program control. Upon reception, a microchip of block507 may utilize a predetermined calibration curve in order to correlatea signal to an accurate ammonia concentration value, as furtherdisclosed in block 508. Block 509 may further disclose a transmission ofdata from the microchip to a display device, which may be eitherphysically or wirelessly connected to microchip, for user accessibility.This method may include the use of fewer or significantly moremicrochips and controllers under additional program control. Furthermicrochips may be useful for various tests, display mechanisms, dataanalysis, and both visual and auditory aesthetics. Microchips may alsofacilitate communication between an exemplary device and an at-homecomputer, cell phone, TV, or other common display and communicationdevices.

FIG. 6 shows an exemplary embodiment of a blood test strip for use withan electronic device further disclosed in FIG. 7. The testing strip maybe large or small in nature, for use in either laboratory settings orpersonal home use. Conduit channel 601 may be the reception point of asample to be tested. A blood droplet, excreted by lancet, may be placedon distal edge of conduit channel 601, where capillary action maytransport sample into sample section 602. Sample section 602 may beU-shaped to increase surface area with a cation exchange membrane 604,such as Nafion. On the opposing side of membrane 604, a reagent section603 may be filled with reagents commonly used with an indophenol orBerthelot's reaction. Bleach, or a similar anion, may be located in aseparated reservoir either on the testing strip or within the electronicdevice in order to ensure the reactivity of certain reagents.

FIG. 7 shows an exemplary embodiment of a testing device under programcontrol and a display device for the presentation of quantitativeanalysis. A blood test strip 701, such as a strip disclosed in FIG. 6,may be inserted into a port or aperture located on testing device 700,and a blood droplet 702 may be dispensed onto a conduit located distallyon blood test strip 701. Upon insertion, an injection mechanism 770 mayeither automatically or manually add bleach or a similar anion to areagent section 703. Bleach, chloramine T, or similar dry or liquidanion may be stored in reservoir 775, and may be refillable as desired.A photodiode or photoresistor 771 may remain inactive for apredetermined period of time until a fill sensor within microchip 773directs the photodiode or photoresister to generate a signalcorresponding to the coloration of reagent section 703. Photodiode orphotoresistor 771 may then alter the current or voltage of the systemwith or without the means of an instrumentational amplifier and emit asignal sent to an analog-to-digital converter 772. Upon conversion to adigital signal, this may be sent to microchip 773 for analysis andfurther program control. Microchip 773 may compute signal and equate toa concentration of ammonium, or specific amino acids, within samplesection 702 by means of pre-programmed calibration curves. Microchip 773may then send data and information to display device 774 for userreadability. Display device 774 may be wholly integrated into testingdevice 700, or may be connected to testing device 700 physically orwirelessly. Additionally, an alternate embodiment of testing device 700may incorporate multiple microchips for further program control, and maybe connected wirelessly or physically to an external display device,such as a computer, cell phone, TV, LCD screen, printer, or similardisplay and communication devices. Testing device 700 may also be incommunication with devices at hospitals or laboratories for ease ofinformation transfer to a user's doctor or medical facility.

FIG. 8 shows the chemical composition of Nafion. Other similar cationexchange membranes or perfluorinated ionomer membranes may also be usedinterchangeably.

The foregoing description and accompanying figures illustrate theprinciples, preferred embodiments and modes of operation of theinvention. However, the invention should not be construed as beinglimited to the particular embodiments or applications discussed above.Additional variations, modifications, and applications of theembodiments discussed above will be appreciated by those skilled in theart. Additional variations and modifications may include, but are notlimited to, the detection of a variety of different amino acids, such asphenylalnine, histidine, tyrosine, glutamate, threonine, serine,leucine, isoleucine, aspartate, valine, glycine, alanine, tryptophan,proline, lysine, arginine, or others. Detection of these amino acids mayinvolve placing dehydrogenase enzymes or other ammonia lyase enzymes inthe sample section of the well, along with the blood, serum, or plasma.Possible applications for the detection of the presence of amino acidsis to diagnose phenylketonuria or other aminoacidopathies oraminoacidemia.

Any and all journal articles, patent applications, issued patents, orother cited references disclosed herein are incorporated by reference intheir respective entireties.

-   PCT Application Serial No. PCT/US2013/065548.-   1. J. Zschocke, G. F. Hoffmann, Vademecum Metabolicum (Milupa    Metabolics, Friedrichsdorf, Germany, ed. 3rd, 2011).-   2. B. C. Lanpher, A. L. Gropman, K. A. Chapman, U.    Lichter-Konecki, M. L. Summar, Urea Cycle Disorders Overview (NCBI    Bookshelf, 2003).-   3. M. L. Summar, S. Koelker, D. Freedenberg, C. Le Mons, J. Haberle,    H.-S. Lee, B. Kirmse, The incidence of urea cycle disorders., Mol.    Genet. Metab. 110, 179-80 (2013).-   4. R. H. Singh, Nutritional management of patients with urea cycle    disorders., J. Inherit. Metab. Dis. 30, 880-7 (2007).-   5. M. Msall, Neurological Outcome in Children with Inborn Errors of    Urea Synthesis.pdf, N. Engl. J. Med. 310, 1500-1505 (1984).-   6. A. L. Gropman, M. L. Batshaw, Cognitive outcome in urea cycle    disorders., Mol. Genet. Metab. 81 Suppl 1, S58-62 (2004).-   7. M. L. Batshaw, S. Brusilow, L. Waber, W. Blom, A. M.    Brubakk, B. K. Burton, H. M. Cann, D. Kerr, P. Mamunes, R.    Matalon, D. Myerberg, I. A. Schafer, Treatment of Inborn Errors of    Urea Synthesis, N. Engl. J. Med. 306, 1387-1392 (1982).-   8. F. F. Poordad, Review article: the burden of hepatic    encephalopathy., Aliment. Pharmacol. Ther. 25 Suppl 1, 3-9 (2007).-   9. R. F. Butterworth, J. F. Giguere, J. Michaud, J. Lavoie, G. P.    Layrargues, Ammonia: key factor in the pathogenesis of hepatic    encephalopathy, Neurochem Pathol 6, 1-12 (1987).-   10. R. F. Butterworth, Pathophysiology of hepatic encephalopathy: a    new look at ammonia., Metab. Brain Dis. 17, 221-7 (2002).-   11. J. Stahl, Studies of the Blood Ammonia in Liver Disease, Ann.    Intern. Med. 58 (1963).-   12. I. Eijgelshoven, S. Demirdas, T. A. Smith, J. M. T. van Loon, S.    Latour, A. M. Bosch, The time consuming nature of phenylketonuria: A    cross-sectional study investigating time burden and costs of    phenylketonuria in the Netherlands, Mol. Genet. Metab. 109, 237-242    (2013).-   13. P. V. D. Burg, H. W. Mook, A simple and rapid method for the    determination of ammonia in blood, Clin. Chim. Acta 8, 162-164    (1962).-   14. Y. Murawaki, K. Tanimoto, C. Hirayama, Y. Ikuta, N. Watabe, A    simple and rapid microdiffusion method for blood ammonia using a    reflectance meter and a reagent plate, and its clinical evaluation    for liver diseases., Clin. Chim. Actal 144 (1984).-   15. R. J. Barsotti, Measurement of ammonia in blood, J. Pediatr.    138, S11-S20 (2001). 16. J. Buttery, R. Ratnaike, B. Chamberlain,    The measurement of erythro-cyte ammonia using the Hyland ammonia    kit, J Clin Chem Clin Biochem 20 (1982).-   17. S. Dienst, An ion exchange method for plasma ammonia    concentration, J. Lab. Clin. Med. 58 (1961).-   18. J. Huizenga, C. Gips, Determination of blood ammonia using the    Ammonia Checker, Ann Clin Biochem 20 (1983).-   19. H. van Anken, M. Schiphorst, A kinetic determination of ammonia    in plasma, Clin Chim Acta 56 (1974).-   20. L. Rover Junior, J. C. Fernandes, G. de Oliveira Neto, L. T.    Kubota, E. Katekawa, S. H. Serrano, Study of NADH stability using    ultraviolet-visible spectrophotometric analysis and factorial    design., Anal. Biochem. 260, 50-5 (1998).-   21. M. Berthelot, B, Repert. Chim. Appl., 254 (1859).-   22. E. D. Rhine, G. K. Sims, R. L. Mulvaney, E. J. Pratt, Improving    the Berthelot Reaction for Determining Ammonium in Soil Extracts and    Water, Soil Sci. Soc. Am. J. 62 (1998).-   23. T. T. Ngo, A. P. H. Phan, C. F. Yam, H. M. Lenhoff, Interference    in Determination of Ammonia with the Hypoehlorite-Alkali Phenol    Method of Berthelot, 46-49 (1981).

The foregoing description and accompanying figures illustrate theprinciples, preferred embodiments and modes of operation of thedisclosure. However, the disclosure should not be construed as beinglimited to the particular embodiments or applications discussed above.Additional variations, modifications, and applications of theembodiments discussed above will be appreciated by those skilled in theart. Additional variations and modifications may include, but are notlimited to, the detection of a variety of different amino acids, such asphenylalnine, histidine, tyrosine, glutamate, threonine, serine,leucine, isoleucine, aspartate, valine, glycine, alanine, tryptophan,proline, lysine, arginine, or others. Detection of these amino acids mayinvolve placing dehydrogenase enzymes or other ammonia lyase enzymes inthe sample section of the well, along with the blood, serum, or plasma.Possible applications for the detection of the presence of ammonia orammonium ion is to diagnose phenylketonuria or other aminoacidopathies.

Therefore, the above-described embodiments should be regarded asillustrative rather than restrictive. Accordingly, it should beappreciated that variations to those embodiments can be made by thoseskilled in the art without departing from the scope of the disclosure asdefined by the following claims.

EXAMPLES Example 1

The presented work demonstrates how the systematic investigation ofpreviously known technologies yielded the fabrication of an effectiveblood ammonia sensor. The indophenol reaction, in tandem with apolyelectrolyte membrane, was explored as a means to quantify ammoniaconcentrations in whole blood.

The ammonia-indophenol standard curve was produced using a range ofammonium chloride concentrations in 1× phosphate buffered saline (PBS)of 0 to 750 μM. The following concentrations were utilized in theindophenol reaction: 59 mM 2-phenylphenol in ethanol, 7 μM sodiumnitroprusside in water, 500 mM sodium hydroxide in water, and 0.2-0.25%aqueous hypochlorite. These concentrations were mixed in a 1:1:1:0.5ratio with an equal volume of the ammonium solution of interest andallowed to react at room temperature for 10 minutes. The absorbance ofthe resulting solution was measured at a wavelength of 635 nm.

Example 2 Stability Studies

The reagents utilized in the indophenol reaction were investigated forlong term stability. Aqueous solutions of hypochlorite, sodiumnitroprusside, sodium hydroxide and a solution of 2-phenylphenol inethanol were stored in separate 50 mL falcon tubes, with limitedexposure to light. At intervals of 3, 5, 7, 15, 21, 28, 35, 50, 75 and100 days the hypochlorite, sodium nitroprusside, sodium hydroxide and2-phenylphenol were utilized to develop a standard curve using ammoniaconcentrations ranging from 0-750 μM. Significant deviations from theoriginal standard curve indicated the degradation of the storedreagents. It should be noted that fresh ammonia samples were utilized ateach test interval.

Response to Amino Acids

Primary amines can also undergo the indophenol reaction. Total aminoacid concentrations in blood can be as high as 2.5 mM, therefore theselectivity of 2-phenylphenol was determined in the indophenol reaction.1 mM solutions of each of the 21 amino acids was prepared in 1×PBS. Thesame protocol utilized with the indophenol reagents for the ammoniastandard curve was utilized with each amino acid solution. 10 minutesafter the indophenol reagents and amino acid solution was mixed, itsabsorbance at 635 nm was measured using a plate reader. The response wasdirectly compared to the response seen from a 1 mM solution of ammoniumchloride and expressed as a percentage of the ammonium response.

Sensor Design

A bisected well containing blood in one section and a concentratedalkali solution in the other would provide a means for cation exchangeof the whole blood to occur, yielding a strong recovery of the ammonium.A computer-aided design of the well that is both reusable and modularwas 3D printed. As seen in FIG. 11, two modular pieces were 3D printedfrom acrylonitrile-butadiene-styrene thermoplastic. The pieces will snaptogether with the membrane in the middle, forming a Nafion bisectedwell. This design was chosen to provide a uniform platform for allfuture experiments involving this sensing mechanism. Silicone gasketingmaterial, at a 1/64″ thickness, was glued to the inner face of eachwell-half to ensure a water tight seal. The wells were then back-filledwith polydimethylsiloxane to improve their mechanical properties.

FIG. 11 Photograph of the 3D printed modular pieces snapped togetheraround Nafion to form the bisected well utilized for the sensingexperiments.

Sensor Response to Ammonia in Phosphate Buffered Saline and Whole Blood

The 3D printed wells were constructed with 1 cm² pieces of Nafionmembrane. In one bisection of the well a range of 0-500 μMconcentrations of ammonium chloride in 1×PBS was added. In the opposingbisection a 1M alkali solution was added. Ion-exchange of ammonium wasallowed to occur for 20 minutes. The alkali solution, now containingammonia, was then extracted and utilized in the indophenol reaction. Theabsorbance of the resulting indophenol reaction was measured at 635 nmafter 10 minutes using a microplate reader.

Whole human blood was spiked using ammonium chloride to generateconcentrations of ammonia of 25, 50, 75, 100, 150, 200, 250, 300, 400and 500 μM. This method of producing ammonia-spiked blood was verifiedutilizing a Siemens RXL to determine the true ammonia concentrations ofthe resulting whole blood. The ammonia-spiked whole blood was pipettedinto the sensor in a protocol identical to the one used in the case ofthe ammonium in 1×PBS. In one section of the well the ammonia-spikedblood was added. In the other section was the concentrated alkalisolution. After 20 minutes the ion-exchange has taken placed and theammonia is extracted into the alkali solution. The ammonia-containingalkali solution was then mixed with the hypochlorite, sodium hydroxide,sodium nitroprusside, and 2-phenylphenol. The resulting indophenolreaction's absorbance was measured at 635 nm after 10 minutes.

Hypochlorite Concentrations Effect on Indophenol Response to BloodAmmonia

To reduce interference from reducing species in blood, higherconcentrations of hypochlorite than conventionally utilized wereemployed in the indophenol reaction with ammonia extracted from wholesheep's blood. 1, 2, 3, 5, and 10× concentrations of hypochlorite wereutilized and the resulting absorbance at 635 nm was recorded.

Ammonia-Indophenol Standard Curve

FIG. 12. The indophenol reaction produces a linear curve withconcentrations of ammonium chloride ranging from 0-750 μM with a COD of0.9939.

The efficacy of the indophenol reaction was initially evaluated for itslower limit of quantification (LLoQ), resolution, range and, responsetime. The utilized reagents were optimized to produce a response from25-1000 μM ammonium chloride demonstrated in FIG. 12. An LLoQ of 25 μMwas recorded with an average error of ˜15%, therefore the sensor'sresolution in terms of concentration is higher at lower ammoniumconcentrations.

Stability Studies

One major advantage of using the indophenol reaction for determiningammonia concentrations is that it does not require any biologicalcomponents such as enzymes, which are prone to stability issues. Theshelf-life of the solutions used for the indophenol reaction wasexamined over the course of 100 days. The components of the indophenolreaction are not stable when mixed together, potentially due to thehypochlorite and the coupling agent's, sodium nitroprusside, reactivity.The response to the range of ammonium chloride concentrations was stablefor up to 50 days. As seen in FIG. 13, the response to 25, 150 and 500μM ammonium chloride does not change significantly until day 75.FIG. 13 The reagents for the indophenol reaction were stored at roomtemperature and used to generate an ammonia standard curve at regularintervals for 100 days. The response to 500 μM ammonia began to degradeat day 75. The reagents of the indophenol reaction are stable at roomtemperature for up to 50 days before its response to differentconcentrations of ammonia begins to deteriorate.

Response to Amino Acids

The mechanism for the indophenol reaction is also applicable to otherprimary amine containing compounds. For whole blood applications this isproblematic due to the presence of small amine containing molecules suchas amino acids which would cause interference when measuring bloodammonia. The phenol compound utilized in the indophenol reaction,2-phenylphenol, is thought to introduce some form of selectivity due thelarge phenyl group adding a degree of steric hindrance to the reaction.The selectivity of the reaction was tested with a large array ofdifferent amino acids. Since the response to the amino acids was so low,a concentration of 500 μM was utilized for ammonia and 1 mM for theamino acids. The absorbance values recorded for the amino acids werenormalized with the ammonia acting as 100%. The radar graph in FIG. 14shows the response of each amino acid, the highest of which wasthreonine that was just 7% of the ammonia response.

FIG. 14 1 mM concentrations of each of the 21 amino were tested usingthe indophenol reaction. The absorbance measured at 635 nm for eachamino acid after the indophenol reaction was calculated as percentage ofthe response from indophenol reaction with 1 mM ammonium chlroide. Theradar graph displays the percent response as compared to ammoniumchloride. The highest response was threonine which produced anabsorbance value that was just 7% of ammonia's response.

Cation Exchange of Whole Blood

The other major source of interference for the indophenol reaction isproteins. Small quantities of proteins can completely disable thereaction from proceeding. In order to rapidly separate ammonium fromwhole blood while excluding any proteins, Nafion, a cation exchangemembrane, was utilized. Nafion has had previous application inbiosensors but almost entirely for protection of electrodes inelectrochemical sensors. In this case Nafion is being operated as acation exchange membrane rather than a nanoporous form of Teflon, asused in electrochemical sensors. Nafion is a fluorinated ionomer blockcopolymer. When cast into films, usually from solution in a hot press,the ionomeric block aggregate into long-range pores of the sulfonessurrounded by a matrix of the fluoropolymer. The pores are highlynegatively charged due to the sulfonic acids groups and are generally1-4 nm in size. These pores allow for the rapid diffusion of hydroxylcontaining molecules and cations through the Nafion while inhibitinganions and completely preventing macromolecules. This would allow forthe rapid diffusion of ammonia while reducing amino acid diffusion andcompletely eliminating proteins from passing and disabling theindophenol reaction.

The ion-exchange of ammonium through the use of the Nafion is the mainmechanism of recovery of the analyte. Ammonium will diffuse across themembrane passively as well, but at a rate that is not sufficient for abeneficial point-of-care sensor. Alkali solutions of different ionicstrength were tested for their effectiveness in exchanging with theammonium from a PBS solution. It was expected that higher concentrationsof salt would yield larger recoveries of ammonium. Bisected wells wereprepared with Nafion membranes. A 500 mM solution of ammonium chloridein PBS was placed on the ‘analyte’ side of the bisected well andsolutions of a concentrated aqueous alkali in the opposing bisection.Distilled water resulted in a 10% recovery, in the control case, whilethe concentrated aqueous alkali resulted in a 75% recovery of theammonium. The larger concentration of ammonium in the alkali solutionversus the analyte is indicative of the ion exchange mechanismoccurring, as the concentration would be equal if the mechanism wassimply passive diffusion.

Sensor Response to Ammonia in PBS

The sensor was initially challenged with ammonium chloride solution inPBS, before introducing an environment as complex as whole blood.Concentrations ranging from 0-500 μM were analyzed. In healthy adultsammonia levels are generally 50-80 μM whereas concentrations greaterthan 100 μM are suspect. These numbers are higher in neonates in whichcase less than 110 μM is normal, up to 180 μM could be attributed toother illnesses and greater than 200 μM is cause for concern. In severecases ammonia levels can be as high as 500 μM(1).FIG. 5 The constructed sensor's response to a range of ammoniaconcentrations in 1×PBS. The COD is 0.9758 with n=5 samples.The sensor reliably extracted the ammonia in 20 minutes time. Theextracted solution was then tested using the indophenol reaction and thedeveloped color analyzed using a plate reader measuring absorbance at635 nm. This process produced the standard curve seen in FIG. 15. TheCOD for detection in PBS was 0.97, with an error of 5-15%. In the rangeof 0-100 mM, there was a resolution of ˜30 μM ammonium. The sensor wasefficacious over the entire clinically relevant range of ammonia levelsin PBS.

Initial Sensor Response to Ammonia in Whole Blood

Initial studies in blood produced a non-linear relationship betweenammonia concentration and absorbance. The response was limited at anabsorbance of 0.35 at a blood ammonia concentration of 500 mM. Theseresponses are markedly reduced from the same concentrations of ammoniain PBS. This suggests that the ammonia is either inhibited fromdiffusing across the Nafion membrane or small molecules from blood areinterfering with the indophenol reaction. Negative interference of theindophenol reaction can also occur from the presence of certain smallmolecules. It has been previously reported that high concentrations ofamines, thiols and reducing agents will disrupt the indophenol reaction,all of which are present in blood(23). Reducing agents will readilyreact with hypochlorite, an oxidizing agent, effectively disabling theindophenol reaction. To determine if this was the case, ammonia extractfrom whole sheep blood was exposed to a modified indophenol reactionusing 2, 3, 5 and 10× more concentrated hypochlorite than conventionallyutilized. As seen in FIG. 17, increasing the hypochlorite concentrationup to 3× improved the reaction's response to the ammonia extracted fromthe whole blood. At 5 and 10× concentrated hypochlorite, the reactionsresponse to ammonia began to degrade, indicating that 3× hypochlorite isoptimal for reducing the interference introduced by reducing agentsfound in blood.

Modified Sensor Response to Ammonia in Whole Blood

The 3× hypochlorite-modified indophenol reaction was examined inconjunction with the Nafion based separation technique for itseffectiveness in distinguishing blood ammonia concentrations rangingfrom 25 to 500 μM, representing healthy to diseased levels. Theresulting standard curve, in FIG. 18, demonstrated the response in thisrange. There was a significant correlation, with a COD of 0.9573,between blood ammonia concentrations and the resulting absorbance at 635nm after the indophenol reaction.

FIG. 18. The bisected well sensor was again used to extract ammonia inwhole human blood. The extracted ammonia solutions were tested with the3× hypochlorite-modifed indophenol reaction and the absorbance measuredat 635 nm. In the range of 0-500 μM the COD was 0.9573 with n=5 samples.

In the range of 25-150 μM, where high resolution measurements arecritical for examining treatment effectiveness, the COD was 0.9777, seenin FIG. 19. The error in this range was ˜10%, giving a preliminaryresolution of 15 μM. The relative standard deviation of 10% percentfalls within the FDA guide for validation of a bioanalytical methodwhich requires an relative standard deviation of 15% at n=5 samples. TheLLoQ, 25 μM, is at least 3σ above the mean background reading of0.04483+/−0.00117 absorbance. Additionally, the sensor can reliablydifferentiate between 50 and 100 μM blood ammonia with a p=0.0001.

FIG. 19 depicts the sensor's response to blood ammonia concentrationsranging from 0-150 μM. The relative standard deviation is ˜10% with aCOD of 0.9777 with n=5 samples.

The investigated bioanalytical method for evaluating blood ammonialevels demonstrated a high degree of correlation between blood ammoniaand sensor response. In the range of from about 25 to about 150 mM, therelative standard deviation was approximately 10%. The sensor has abouta twenty-minute response time, and the interference from other smallmolecules was greatly reduced. The components used are stable at roomtemperature for up 50 days and inexpensive.

Validation of the functionality of the biosensor will also be performedusing the following experimental design. The evaluation of the efficacyof the phenylalanine sensor requires the construction of a three carbonelectrode modified with an alginate hydrogel consisting of alginate,CaCl2, Toluidine Blue, Phenylalanine Dehydrogenase, and NAD(P)+. Thehydrogel will act as a filter to prevent interference from smallmolecules and proteins in whole blood. Initially 32 whole blood sampleswill be tested with phenylalanine concentrations ranging from about 35μM to about 2000 μM. Specifically the following Phe concentrations willbe tested on the enzyme electrode: 35, 100, 250, 500, 1000, 1250, 1500,2000 μM. In this experiment 35 μM will represent a physiologicallynormal concentration and each other concentration above 100 μM willrepresent a variety of different diseased concentrations. Theseconcentrations will be generated by doping whole blood of aconcentration lower than 35 μM. Additionally, subject samples will betested to ensure the sensor operates without issues form unforeseenabnormalities with patient whole blood. All phenylalanine concentrationwill be verified by the use of high performance liquid chromatography(HPLC), which is the gold standard for determining phenylalanine levelsin blood. The samples will not require preprocessing. The blood will betaken using sodium heparin vacuum tubes and then used unmodified outsideof doping the blood with higher phenylalanine concentrations. Theexpected detection limit is 35 μM with a range of 35-2000 μM and aresolution of 20 μM. Statistical Evaluations will be performed to assessthe reliability of the concentration measurements. The concentrationmeasurements will be analyzed using ANOVA single factor analysis todemonstrate differences between groups assuming a normal datadistribution. Confidence intervals will be assessed and the sensitivityand the reproducibility of the method demonstrated. Concentrationmeasurements ranging from normal physiological conditions to diseasedconditions with a confidence 95% (p<0.05) or higher will be deemedstatistically significant. Statistical differences between diseasedconcentration levels and healthy, physiological concentration levelswill first be demonstrated. Subsequent experimentation will be used tovalidate quantification of over the full range of discrete concentrationvalues.

Example 3

Measuring Ammonia in Whole Blood with a Modular Well Plate

Materials

2-phenylphenol, sodium nitroprusside, sodium hydroxide, sodiumhypochlorite, sodium acetate, and ammonium chloride were purchased fromSigma-Aldrich. Nafion 111 was purchased from Ion-Power. 1/64″ siliconegasket with adhesive backing was purchased from McMaster-Carr,Acrylonitrile Butadiene Styrene Resin.

Methods

Preparation of Sample

-   -   Prepare stock solutions of 59 mM 2-phenylphenol in ethanol, 7 μM        sodium nitroprusside in water, 500 mM sodium hydroxide in water        and 0.6-0.75% sodium hypochlorite in water.    -   Prepare a stock solution of 1M sodium acetate in water.    -   3D printed wells are produced from fused deposition modeling        using acrylonitrile butadiene styrene based on the model seen in        FIG. 210.    -   Attach a layer of the 1/64″ silicone to the area of the well        shaded in FIG. 21.    -   Remove the 25 μm thick Nafion 111 from plastic backing and cut        it into 1.5×1.5 cm squares.

Ammonia Exchange

-   -   Snap two of the modular 3D printed pieces around one of the        Nafion Squares, creating a Bisected Well. The final        configuration is shown in FIG. 22.    -   Pipette 45 μl of the sodium acetate solution into section B of        the well.    -   Pipette 100 μM of the ammonia containing sample (whole blood)        into section A of the well.    -   Wait 20 minutes for ion exchange to occur.

Collection of Data

-   -   Pipette 35 μl of the sodium acetate/ammonia solution from        section B into a 384 well plate.    -   Pipette 10 μl each of the 2-phenylphenol, sodium nitroprusside        and sodium. hydroxide stock solutions into the same well as the        sodium acetate.    -   Add 5 microliters of the sodium hypochlorite solution to the        well and mix thoroughly by pipette.    -   Wait ten minutes for the indophenol reaction to proceed.    -   Measure the absorbance of the well at 635 nm using a 384 plate        reader.    -   Compare the measured absorbance to a standard curve to determine        unknown ammonia concentration.        FIG. 20 CAD sketch of the front face of the well plate.        FIG. 21 CAD sketch demonstrating the area of the well plate that        the adhesive silicone should be attached to (black area).        FIG. 22 Photograph of the 3D printed modular pieces snapped        together around Nafion to divide the well into two sections.        FIG. 23 Engineering Drawing of 3D printed well.

Example 4

Measuring Ammonia in Whole Blood with a Fluidic Device

Materials

2-phenylphenol, sodium nitroprusside, sodium hydroxide, sodiumhypochlorite, sodium acetate, and ammonium chloride were purchased fromSigma-Aldrich. Nafion 111 was purchased from Ion-Power.

Methods Preparation of Sample

-   -   Prepare stock solutions of 59 mM 2-phenylphenol in ethanol, 7 μM        sodium nitroprusside in water, 500 mM sodium hydroxide in water        and 0.75% sodium hypochlorite in water.    -   Prepare a stock solution of 1M sodium acetate in water.    -   Molds are produced from a 3D printer for the 2 individual pieces        of the device.    -   Fill each mold with the PDMS elastomer and heat at 60° C. for        one hour.    -   Remove each side of the device from the mold with a spatula.    -   Remove the 25 μm thick Nafion 111 from plastic backing and cut        it into a 1.5×1.5 cm square.    -   Glue the square of Nafion over the well in the channel 6 using        PDMS.    -   Glue the top piece of the device to the bottom piece using PDMS,        ensuring channel 6 lines up with channel 5.    -   Heat the device to 60° C. for one hour.

Ammonia Exchange

-   -   Insert a needle through the bottom of the device into well 6,        and fill with 40 μl of blood.    -   Fill channels 1-4 with 5 μl of 2-phenylphenol, sodium        nitroprusside, sodium hydroxide and sodium hypchlorite        respectively.    -   Fill channel 5 with 20 μl of sodium acetate solution.    -   Wait 20 minutes for ion exchange to occur.

Collection of Data

-   -   Apply a flow rate of 1 mm/sec to channels 1 through 5 for 24        seconds.    -   Wait ten minutes for the indophenol reaction to proceed.    -   Insert the device into the custom photo-spectrometer to acquire        absorbance data.    -   Compare the measured absorbance to a standard curve to determine        unknown ammonia concentration.

An embodiment depicting a method of detecting amino acids based uponammonia levels is depicted in FIG. 29. After addition of blood asdepicted on the left handside of FIG. 29, ammonia levels are visualizedby a color change of ammonia reaction with indophenol reagents in theleft well of the device depicted on the righthand side. The left well ismore grey (an indication of blue color) than the left well in the devicedepicte on the lefthand side (before the indophenol reaction takesplace).

Example 5 Dection of Amino Acids in Whole Blood Through Use of AmmoniaProducing Enzymes with a Modular Well Plate

Dehydrogenases and ammonia-lyases generally affect amino acids bycleaving off the primary amine thereby generating ammonia. Using theammonia dection modular well plate system described herein, the ammoniagenerated, measured via the color change produced, can then becorrelated to the concentration of a given amino acid. This exampletests for the presence of phenylaline using phenylalanine ammonia-lyase,although other amino acids can be tested by using the appropriateenzyme(s) listed on Table 5.

TABLE 5 Phenylalnine Histidine Tyrosine Glutamate Threonine SerinePhenylalanine Histidine Tyrosine Glutamate Threonine Serine AmmoniaAmmonia Ammonia Dehydrogenase Ammonia Ammonia Lyase, Lyase Lyase Lyase,Lyase, Serine Phenylalanine Threonine Dehydrogenase DehydrogenaseDehydrogenase Leucine Isoleucine Aspartate Valine Glycine AlanineLeucine Isoleucine Aspartate Valine Glycine Alanine DehydrogenaseDehydrogenase Ammonia Dehydrogenase Dehydrogenase Dehydrogenase Lyase,Aspartate Dehydrogenase Tryptophan Proline Lysine Arginine TryptophanProline Lysine Arginine Dehydrogenase Dehydrogenase DehydrogenaseDehydrogenase

Materials

2-phenylphenol, sodium nitroprusside, sodium hydroxide, sodiumhypochlorite, sodium acetate, and ammonium chloride were purchased fromSigma-Aldrich. Nafion 111 was purchased from Ion-Power. 1/64″ siliconegasket with adhesive backing was purchased from McMaster-Carr,Acrylonitrile Butadiene Styrene Resin, phenylalanine ammonia-lyase,sodium alginate from brown algae (optional), phosphate buffered saline(optional), 0.1M CaCl2 solution (optional).

Methods

Preparation of Sample

-   -   Prepare stock solutions of 59 mM 2-phenylphenol in ethanol, 7 μM        sodium nitroprusside in water, 500 mM sodium hydroxide in water        and 0.6-0.75% sodium hypochlorite in water.    -   Prepare a stock solution of 1M sodium acetate in water.    -   3D printed wells are produced from fused deposition modeling        using acrylonitrile butadiene styrene based on the model seen in        FIG. 210.    -   Attach a layer of the 1/64″ silicone to the area of the well        shaded in FIG. 21.    -   Remove the 25 μm thick Nafion 111 from plastic backing and cut        it into 1.5×1.5 cm squares.

The enzyme can be added to the amino acid containg sample (whole blood)in two ways. Either by adding in the enzyme directly to the sample, orby immobilizing the enzyme in a gel placed inside the sample well,introducing greater enzyme stability.

Ammonia Exchange with Free Enzyme (Option 1)

-   -   Snap two of the modular 3D printed pieces around one of the        Nafion Squares, creating a Bisected Well. The final        configuration is shown in FIG. 22.    -   Pipette 45 μl of the sodium acetate solution into section B of        the well.    -   Pipette 100 μM of the amino acid containing sample (whole blood)        into section A of the well.    -   Add 40 units of phenylalanine ammonia-lyase to the sample.    -   Wait 20 minutes for ion exchange to occur.

Ammonia Exchange with Gel-Immobilized Enzyme (Option 2)

-   -   Snap two of the modular 3D printed pieces around one of the        Nafion Squares, creating a Bisected Well. The final        configuration is shown in FIG. 22.    -   Prepare a pre-gel solution of 40 units of phenylalanine        ammonia-lyase, and 1% weight/volume sodium alginate from brown        algae in 1 mL 1× phosphate buffered saline.    -   Place 10 μL of the pre-gel solution into section A of the well        (where the whole blood will go).    -   Spray the pre-gel solution in the well with 0.1M CaCl2 solution        using a Badger 200N airbrush at 7.5 psi for 1 second, depositing        ˜5 μL of the CaCl2 solution. The gel will be allowed to cure for        30 minutes in a humid environment.    -   Pipette 45 μl of the sodium acetate solution into section B of        the well.    -   Pipette 100 μM of the amino acid containing sample (whole blood)        into section A of the well.    -   Wait 20 minutes for ion exchange to occur.

Collection of Data

-   -   Pipette 35 μl of the sodium acetate/ammonia solution from        section B into a 384 well plate.    -   Pipette 10 μl each of the 2-phenylphenol, sodium nitroprusside        and sodium. hydroxide stock solutions into the same well as the        sodium acetate.    -   Add 5 microliters of the sodium hypochlorite solution to the        well and mix thoroughly by pipette.    -   Wait ten minutes for the indophenol reaction to proceed.    -   Measure the absorbance of the well at 635 nm using a 384 plate        reader.    -   Compare the measured absorbance to a standard curve to determine        unknown ammonia concentration.

Example 6 Dection of Amino Acids in Whole Blood Through Use of AmmoniaProducing Enzymes with a Fluidic Device

Dehydrogenases and ammonia-lyases generally affect amino acids bycleaving off the primary amine thereby generating ammonia. Using theammonia dection modular well plate system described herein, the ammoniagenerated, measured via the color change produced, can then becorrelated to the concentration of a given amino acid. This exampletests for the presence of phenylaline using phenylalanine ammonia-lyase,although other amino acids can be tested by using the appropriateenzyme(s) listed on Table 5.

Materials

2-phenylphenol, sodium nitroprusside, sodium hydroxide, sodiumhypochlorite, sodium acetate, and ammonium chloride were purchased fromSigma-Aldrich. Nafion 111 was purchased from Ion-Power, phenylalanineammonia-lyase, sodium alginate from brown algae (optional), phosphatebuffered saline (optional), 0.1M CaCl2 solution (optional).

Methods

Preparation of Sample

-   -   Prepare stock solutions of 59 mM 2-phenylphenol in ethanol, 7 μM        sodium nitroprusside in water, 500 mM sodium hydroxide in water        and 0.75% sodium hypochlorite in water.    -   Prepare a stock solution of 1M sodium acetate in water.    -   Molds are produced from a 3D printer for the 2 individual pieces        of the device.    -   Fill each mold with the PDMS elastomer and heat at 60° C. for        one hour.    -   Remove each side of the device from the mold with a spatula.    -   Remove the 25 μm thick Nafion 111 from plastic backing and cut        it into a 1.5×1.5 cm square.    -   Glue the square of Nafion over the well in the channel 6 using        PDMS.    -   Glue the top piece of the device to the bottom piece using PDMS,        ensuring channel 6 lines up with channel 5.    -   Heat the device to 60° C. for one hour.

The enzyme can be added to the amino acid containg sample (whole blood)in two ways. Either by adding in the enzyme directly to the sample, orby immobilizing the enzyme in a gel placed inside the sample well,introducing greater enzyme stability.

Ammonia Exchange with Free Enzyme (Option 1)

-   -   Insert a needle through the bottom of the device into well 6,        and fill with 40 μl of blood.    -   Add 40 units of phenylaline ammonia-lyase to the blood sample.    -   Fill channels 1-4 with 5 μl of 2-phenylphenol, sodium        nitroprusside, sodium hydroxide and sodium hypchlorite        respectively.    -   Fill channel 5 with 20 μl of sodium acetate solution.    -   Wait 20 minutes for ion exchange to occur.

Ammonia Exchange with Gel-Immobilized Enzyme (Option 2)

-   -   Prepare a pre-gel solution of 40 units of phenylalanine        ammonia-lyase, and 1% weight/volume sodium alginate from brown        algae in 1 mL 1× phosphate buffered saline.    -   Place 10 μL of the pre-gel solution into section A of the well        (where the whole blood will go).    -   Spray the pre-gel solution in the well with 0.1M CaCl2 solution        using a Badger 200N airbrush at 7.5 psi for 1 second, depositing        ˜5 μL of the CaCl2 solution. The gel will be allowed to cure for        30 minutes in a humid environment.    -   Insert a needle through the bottom of the device into well 6,        and fill with 40 μl of blood.    -   Add 40 units of phenylaline ammonia-lyase to the blood sample.    -   Fill channels 1-4 with 5 μl of 2-phenylphenol, sodium        nitroprusside, sodium hydroxide and sodium hypchlorite        respectively.    -   Fill channel 5 with 20 μl of sodium acetate solution.    -   Wait 20 minutes for ion exchange to occur.

Collection of Data

-   -   Apply a flow rate of 1 mm/sec to channels 1 through 5 for 24        seconds.    -   Wait ten minutes for the indophenol reaction to proceed.    -   Insert the device into the custom photo-spectrometer to acquire        absorbance data.    -   Compare the measured absorbance to a standard curve to determine        unknown ammonia concentration.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the disclosure described herein. The scope of the presentdisclosure is not intended to be limited to the above Description, butrather is as set forth in the following claims:

1.-47. (canceled)
 48. A method of diagnosing a metabolic disease in asubject comprising: (a) contacting a sample of bodily fluid to abiosensor, wherein the biosensor comprises: (i) at least a first andsecond vessel; (ii) a fluid exchange opening positioned between thefirst and the second vessel; (iii) at least one conduit in fluidcommunication with the at least first vessel, the at least one conduitconfigured to receive a fluid from a point external to the biosensor;and (iv) a membrane positioned at the fluid exchange opening; whereinthe membrane comprises an ionomer; wherein the at least one conduit isconfigured to hold a sample volume from about 5 μl to about 100 μl; andwherein the first vessel or the second vessel comprises, individually orin combination: a hypohalite, an alkali buffer, and at least onephenolic reagent or indophenol related compound; (b) quantifying one ormore concentration values of ammonia in the sample; (c) comparing theone or more concentration values of ammonia in the sample to a thresholdvalue of ammonia concentration identified as being in a healthy range;and (d) identifying the subject as having a metabolic disease if the oneor more concentration values of ammonia in the sample exceed or fallbelow the threshold value.
 49. The method of claim 48, wherein themetabolic disease is hyperammonemia.
 50. The method of claim 48, whereinthe method does not comprise exposing the sample of bodily fluid to anyexternal stimuli or reagent prior to contacting the sample to the biosensor.
 51. The method of claim 48, wherein the sample of bodily fluidis whole blood from a subject.
 52. The method of claim 48, wherein thesample of bodily fluid does not contain urine.
 53. The method of claim48, wherein the sample of bodily fluid is in contact with the biosensorfor a sufficient period of time to allow modification of the phenolicreagent or indophenol related compound to become modified into aphenolic or indophenol reaction product.
 54. The method of claim 53,wherein the sample of bodily fluid is in contact with the biosensor forno more than 20 minutes.
 55. The method of claim 48, wherein the firstvessel and/or or the second vessel comprises a catalyst.
 56. A method ofdetecting the presence, absence, or quantity of amino acid in a samplecomprising: (a) contacting a sample of bodily fluid to a biosensor,wherein the biosensor comprises: (i) at least a first and second vessel;(ii) a fluid exchange opening positioned between the first and thesecond vessel; (iii) at least one conduit in fluid communication withthe at least first vessel, the at least one conduit configured toreceive a fluid from a point external to the biosensor; and (iv) amembrane positioned at the fluid exchange opening; wherein the membranecomprises an ionomer; wherein the at least one conduit is configured tohold a sample volume from about 5 μl to about 100 μl; and wherein thefirst vessel or the second vessel comprises, individually or incombination: a hypohalite, an alkali buffer, and at least one phenolicreagent or indophenol related compound; (b) quantifying one or moreammonia or ammonium ion concentration values; and (c) correlating theone or more ammonia or ammonium ion concentration values to one or morequantities of an amino acid.
 57. The method of claim 56, wherein themethod does not comprise exposing the sample of bodily fluid to anyexternal stimuli or reagent prior to contacting the sample to the biosensor.
 58. The method of claim 56, wherein the sample of bodily fluidis whole blood from a subject.
 59. The method of claim 56, wherein thesample of bodily fluid does not contain urine.
 60. The method of claim56, wherein the sample of bodily fluid is in contact with the biosensorfor a sufficient period of time to allow modification of the phenolicreagent or indophenol related compound to become modified into aphenolic or indophenol reaction product.
 61. The method of claim 13,wherein the sample of bodily fluid is in contact with the biosensor forno more than 20 minutes.
 62. A method of quantifying a concentration ofammonia or ammonium ion in a sample comprising contacting a sample ofbodily fluid to a biosensor, wherein the biosensor comprises: (i) atleast a first and second vessel; (ii) a fluid exchange openingpositioned between the first and the second vessel; (iii) at least oneconduit in fluid communication with the at least first vessel, the atleast one conduit configured to receive a fluid from a point external tothe biosensor; and (iv) a membrane positioned at the fluid exchangeopening; wherein the membrane comprises an ionomer; wherein the at leastone conduit is configured to hold a sample volume from about 5 μl toabout 100 μl; and wherein the first vessel or the second vesselcomprises, individually or in combination: a hypohalite, an alkalibuffer, and at least one phenolic reagent or indophenol relatedcompound; wherein the sample is in contact with the biosensor for asufficient period of time to allow modification of the phenolic reagentor indophenol related compound to become modified into a phenolic orindophenol reaction product.
 63. The method of claim 62, wherein themethod does not comprise exposing the sample of bodily fluid to anyexternal stimuli or reagent prior to contacting the sample to the biosensor.
 64. The method of claim 62, wherein the sample of bodily fluidis whole blood from a subject.
 65. The method of claim 62, furthercomprising comparing a concentration value obtained by the contactingstep to a threshold value associated with one or more metabolicdiseases.
 66. The method of claim 65, wherein the metabolic disease ishyperammonemia.
 67. A method of manufacturing a biosensor, comprisingaffixing a membrane between a first and second vessel, wherein thebiosensor comprises: (i) at least the first and second vessel; (ii) afluid exchange opening positioned between the first and the secondvessel; (iii) at least one conduit in fluid communication with the atleast first vessel, the at least one conduit configured to receive afluid from a point external to the biosensor; and (iv) the membranepositioned at the fluid exchange opening; wherein the membrane comprisesan ionomer; wherein the at least one conduit is configured to hold asample volume from about 5 μl to about 100 μl; and wherein the firstvessel or the second vessel comprises, individually or in combination: ahypohalite, an alkali buffer, and at least one phenolic reagent orindophenol related compound.